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Multiple membrane organelles require cholesterol for proper function within cells. The Niemann-Pick type C (NPC) proteins export cholesterol from endosomes to other membrane compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), trans-Golgi network (TGN), and mitochondria, to meet their cholesterol requirements. Defects in NPC cause malfunctions in multiple membrane organelles and lead to an incurable neurological disorder. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), a resident enzyme in the ER, converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs in an NPC-independent manner. Here we report the interesting finding that in a mutant Npc1 mouse (Npc1nmf), Acat1 gene (Soat1) knockout delayed the onset of weight loss, motor impairment, and Purkinje neuron death. It also improved hepatosplenic pathology and prolonged lifespan by 34%. In mutant NPC1 fibroblasts, ACAT1 blockade (A1B) increased cholesterol content associated with TGN-rich membranes and mitochondria, while decreased cholesterol content associated with late endosomes. A1B also restored proper localization of syntaxin 6 and golgin 97 (key proteins in membrane trafficking at TGN) and improved the levels of cathepsin D (a key protease in lysosome and requires Golgi/endosome transport for maturation) and ABCA1 (a key protein controlling cholesterol release at PM). This work supports the hypothesis that diverting cholesterol from storage can benefit multiple diseases that involve cholesterol deficiencies in cell membranes.

While homozygous pathogenic mutations in the NPC1 gene cause Niemann-Pick type C1 disease, heterozygous mutations cause highly-penetrant obesity. We aimed to investigate the prevalence of NPC1 mutations and their signatures of natural selection in 122,678 exome sequenced participants from six ethnic groups in the Genome Aggregation Database. Pathogenic missense coding mutations were identified by in silico tools and the ClinVar database. Signatures of natural selection were assessed by the probability of NPC1 being loss-of-function mutation intolerant and Z-scores of observed/expected synonymous and non-synonymous mutation ratios. There was no evidence of negative selection observed for synonymous, non-synonymous and loss-of-function mutations. However, there were significant ethnic differences in the prevalence of heterozygous pathogenic NPC1 mutations ranging from 0.56% in Ashkenazi Jewish to 3.26% in African/African Americans (5.8-fold difference). Four homozygous carriers of pathogenic NPC1 mutations were also identified, belonging to the South Asian population. In conclusion, NPC1 mutations are consistent with a model of balanced selection, where heterozygotes and homozygotes have higher and lower reproductive fitness, respectively. Therefore, NPC1 heterozygous mutations may account for a substantial and ethnic-dependent percentage of obesity in the general population, while NPC1 homozygous mutations may be frequent in the South Asian populations and warrants more investigation.

Miglustat has been indicated for the treatment of Niemann-Pick disease type C (NP-C) since 2009. The aim of this observational study was to assess the effect of miglustat on long-term survival of patients with NP-C. Data for 789 patients from five large national cohorts and from the NPC Registry were collected and combined. Miglustat-treated and untreated patients overall and within sub-groups according to age-at-neurological-onset, that is, early infantile-onset (<2 years), late infantile-onset (2 to <6 years), juvenile-onset (6 to <15 years), and adolescent/adult-onset (≥15 years) were analysed and compared. Survival was analysed from the time of first neurological manifestation (Neurological onset group, comprising 669 patients) and from diagnosis (Diagnosis group, comprising 590 patients) using a Cox proportional hazard model adjusted for various covariates. Overall, 384 (57.4%) patients in the Neurological onset group and 329 (55.8%) in the Diagnosis group were treated with miglustat. Miglustat treatment was associated with a significant reduction in risk of mortality in both groups (entire Neurological onset group, Hazard ratio [HR] = 0.51; entire Diagnosis group, HR = 0.44; both P < .001). The effect was observed consistently in all age-at-neurological-onset sub-groups (HRs = 0.3 to 0.7) and was statistically significant for late infantile-onset patients in both groups (Neurological onset group, HR = 0.36, P < .05; Diagnosis group, HR = 0.32, P < .01), and juvenile-onset patients in the Diagnosis group only (HR = 0.30, P < .05). Despite the limitations of the data that urge cautious interpretation, the findings are consistent with a beneficial effect of miglustat on survival in patients with NP-C., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Niemann-Pick disease type C1 (NPC1) is a rare, fatal neurodegenerative disorder characterized by lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. NPC1 is extremely heterogeneous in the timing of clinical presentation and is associated with a wide spectrum of causative NPC1 mutations. To study the genetic architecture of NPC1, we have generated a new NPC1 mouse model, Npc1 em1Pav Npc1 em1Pav/em1Pav mutants showed notably reduced NPC1 protein compared to controls and displayed the pathological and biochemical hallmarks of NPC1. Interestingly, Npc1 em1Pav/em1Pav mutants on a C57BL/6J genetic background showed more severe visceral pathology and a significantly shorter lifespan compared to Npc1 em1Pav/em1Pav mutants on a BALB/cJ background, suggesting that strain-specific modifiers contribute to disease severity and survival. QTL analysis for lifespan of 202 backcross N2 mutants on a mixed C57BL/6J and BALB/cJ background detected significant linkage to markers on chromosomes 1 and 7. The discovery of these modifier regions demonstrates that mouse models are powerful tools for analyzing the genetics underlying rare human diseases, which can be used to improve understanding of the variability in NPC1 phenotypes and advance options for patient diagnosis and therapy.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease follows a slow and silent progression, and the transition from early-stage lesions to vulnerable plaques remains difficult to diagnose. Inflammation is a key component of the development of atherosclerotic plaque and consequent life-threatening complications. This study assessed 111 In-DANBIRT as an in vivo , noninvasive SPECT/CT imaging probe targeting an inflammatory marker, Lymphocyte Function Associated Antigen-1 (LFA-1), in atherosclerotic plaques. Methods . Selective binding of 111 In-DANBIRT was assessed using Sprague-Dawley rats exposed to filtered air and ozone (1 ppm) by inhalation for 4 hours to induce a circulating leukocytosis and neutrophilia in peripheral blood. After 24 hours, whole blood was collected and incubated with radiolabeled DANBIRT ( 68 Ga-DANBIRT and 111 In-DANBIRT). Isolated cell component smeared slides using cytospin technique were stained with Wright-Giemsa stain. Apolipoprotein E-deficient (apoE -/- ) mice were fed either a normal diet or a high-fat diet (HFD) for 8 weeks. Longitudinal SPECT/CT imaging was performed 3 hours after administration at baseline, 4, and 8 weeks of HFD diet, followed by tissue harvesting for biodistribution, serum lipid analysis, and histology. 3D autoradiography was performed in both groups 24 hours after administration of 111 In-DANBIRT. Results . Increased specific uptake of radiolabeled DANBIRT by neutrophils in the ozone-exposed group was evidenced by the acute immune response due to 4-hour ozone exposure. Molecular imaging performed at 3 hours using SPECT/CT imaging evidenced an exponential longitudinal increase in 111 In-DANBIRT uptake in atherosclerosis lesions in HFD-fed mice compared to normal-diet-fed mice. Such results were consistent with increased immune response to vascular injury in cardiovascular and also immune tissues, correlated by 24 hours after administration of 3D autoradiography. Histologic analysis confirmed atherosclerotic disease progression with an increased vascular lesion area in HFD-fed mice compared to normal-diet-fed mice. Conclusion . 111 In-DANBIRT is a promising molecular imaging probe to assess inflammation in evolving atheroma and atherosclerotic plaque.

The Niemann-Pick type C1 (NPC1) protein regulates the transport of cholesterol and fatty acids from late endosomes/lysosomes and has a central role in maintaining lipid homeostasis. NPC1 loss-of-function mutations in humans cause NPC1 disease, a rare autosomal-recessive lipid-storage disorder characterized by progressive and lethal neurodegeneration, as well as liver and lung failure, due to cholesterol infiltration. In humans, genome-wide association studies and post-genome-wide association studies highlight the implication of common variants in NPC1 in adult-onset obesity, body fat mass, and type 2 diabetes. Heterozygous human carriers of rare loss-of-function coding variants in NPC1 display an increased risk of morbid adult obesity. These associations have been confirmed in mice models, showing an important interaction with high-fat diet. In this review, we describe the current state of knowledge for NPC1 variants in relationship to pleiotropic effects on metabolism. We provide evidence that NPC1 gene variations may predispose to common metabolic diseases by modulating steroid hormone synthesis and/or lipid homeostasis. We also propose several important directions of research to further define the complex roles of NPC1 in metabolism. This review emphasizes the contribution of NPC1 to obesity and its metabolic complications.

A large number of genome-wide association studies, transferability studies, and candidate gene studies performed in diverse populations around the world have identified gene variants that are associated with common human obesity. The mounting evidence suggests that these obesity gene variants interact with multiple environmental factors and increase susceptibility to this complex metabolic disease. The objective of this review article is to provide concise and updated information on energy balance, heritability of body weight, origins of gene variants, and gene-nutrient interactions in relation to human obesity. It is proposed that knowledge of these related topics will provide valuable insight for future preventative lifestyle intervention using targeted nutritional and medicinal therapies.

It is generally accepted that the selection of gene variants during human evolution optimized energy metabolism that now interacts with our obesogenic environment to increase the prevalence of obesity. The purpose of this study was to perform a global evolutionary and metabolic analysis of human obesity gene risk variants (110 human obesity genes with 127 nearest gene risk variants) identified using genome-wide association studies (GWAS) to enhance our knowledge of early and late genotypes. As a result of determining the mean frequency of these obesity gene risk variants in 13 available populations from around the world our results provide evidence for the early selection of ancestral risk variants (defined as selection before migration from Africa) and late selection of derived risk variants (defined as selection after migration from Africa). Our results also provide novel information for association of these obesity genes or encoded proteins with diverse metabolic pathways and other human diseases. The overall results indicate a significant differential evolutionary pattern for the selection of obesity gene ancestral and derived risk variants proposed to optimize energy metabolism in varying global environments and complex association with metabolic pathways and other human diseases. These results are consistent with obesity genes that encode proteins possessing a fundamental role in maintaining energy metabolism and survival during the course of human evolution., (Copyright © 2017. Published by Elsevier B.V.)

A genome-wide association study (GWAS) reported that common variation in the human Niemann-Pick C1 gene ( NPC1 ) is associated with morbid adult obesity. This study was confirmed using our BALB/cJ Npc1 mouse model, whereby heterozygous mice ( Npc1 +/- ) with decreased gene dosage were susceptible to weight gain when fed a high-fat diet (HFD) compared with homozygous normal mice ( Npc1 +/+ ) fed the same diet. The objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. Metabolic phenotype analysis indicated no significant difference between Npc1 +/+ and Npc1 +/- mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption. However, the livers from Npc1 +/- mice had significantly increased amounts of mature sterol regulatory element-binding protein-1 (SREBP-1) and increased expression of SREBP-1 target genes that regulate glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1 +/- mice had significantly decreased amounts of phosphorylated hormone-sensitive lipase with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1 +/- fibroblasts had significantly increased glycolysis and lipogenesis, in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways., (Copyright © 2017 the American Physiological Society.)

Objective: Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) that increase the risk of developing non-alcoholic fatty liver disease (NAFLD). One purpose of this study was to determine the frequencies of NAFLD susceptibility SNPs in a non-Hispanic white and Hispanic population who attended a clinic in northeast Albuquerque, NM. Another goal was to determine associations with selected indicators in this New Mexican population., Methods: This cohort study involving 168 volunteer subjects in the NM population (88 non-Hispanic whites, 63 Hispanics, 4 Native Americans, 11 Asian Americans, 2 unreported ethnicity). Eight SNPs within 6 NAFLD susceptibility genes including PNPLA3 (rs738409), LYPLAL1 (rs12137855), APOC3 (rs2854116, rs2854117), GCKR (rs780094, rs741038), FABP2 (rs1799883), PEMT (rs7946) were analyzed by genotyping using the TaqMan genotyping assay (Applied Biosystems, Foster City, CA). Statistical analyses were carried out using statistical package SAS 9.3., Results: The NAFLD allele frequencies were similar in non-Hispanic whites and Hispanics except for PNPLA3 (rs738409), FABP2 (rs1799883), and PEMT (rs7946). Eight SNPs in 5 NAFLD susceptibility genes were significantly associated OR marginally associated with selected indicators for NAFLD, metabolic syndrome, overweight, obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia. No SNPs were significantly associated with the same indicator in both the non-Hispanic white and Hispanic groups., Conclusions: In this population of non-Hispanic whites and Hispanics, there were only heterozygotes for the APOC3 derived alle le whereas for all other genes tested, both heterozygotes and homozygotes were found. Associations of alleles with indicators of chronic disease were different in non-Hispanic whites compared to Hispanics., Competing Interests: Conflict of Interest: The authors declare no conflict of interest.

A genome-wide association study (GWAS) and subsequent replication studies in diverse ethnic groups indicate that common Niemann-Pick C1 gene ( NPC1 ) polymorphisms are associated with morbid-adult obesity or diabetes independent of body weight. The objectives for this prospective cross-sectional study were to determine allele frequencies for NPC1 polymorphisms (644A>G, 1926C>G, 2572A>G, and 3797G>A) and association with metabolic disease phenotypes in an ethnically diverse New Mexican obstetric population. Allele frequencies for 1926C>G, 2572A>G, and 3797G>A were significantly different between race/ethnic groups (non-Hispanic white, Hispanic, and Native American). The results also indicated a significant pairwise linkage-disequilibrium between each of the four NPC1 polymorphisms in race/ethnic groups. Moreover, the derived and major allele for 1926C>G was associated (OR 2.11, 95% CI 1.10-3.96, P = 0.022) with increased risk for maternal prepregnancy overweight (BMI 25.0-29.9kg/m 2 ) while the ancestral and major allele for 2572A>G was associated (OR 4.68, 95% CI 1.23-17.8, P = 0.024) with increased risk for gestational diabetes in non-Hispanic whites, but not Hispanics or Native Americans. In summary, this is the first transferability study to investigate common NPC1 polymorphisms in a multiethnic population and demonstrate a differential association with increased risk for maternal prepregnancy overweight and gestational diabetes.

The Niemann-Pick C1 (NPC1) protein has a central role in regulating the efflux of lipoprotein-derived cholesterol from late endosomes/lysosomes and transport to other cellular compartments. Since the NPC1 protein has been shown to regulate the transport of cholesterol to cellular compartments enriched with the ubiquitous cholesterol-binding and transport protein caveolin-1, the present study was performed to determine whether the NPC1 and caveolin-1 proteins interact and function to modulate efflux of low density lipoprotein (LDL)-derived cholesterol from endocytic compartments. To perform these studies, normal human fibroblasts were grown in media with lipoprotein-deficient serum (LPDS) or media with LPDS supplemented with purified human LDL. The results indicated reciprocal co-immunoprecipitation and partial co-localization of the NPC1 and caveolin-1 proteins that was decreased when fibroblasts were grown in media with LDL. Consistent with interaction of the NPC1 and caveolin-1 proteins, a highly conserved caveolin-binding motif was identified within a cytoplasmic loop located adjacent to the sterol-sensing domain (SSD) of the NPC1 protein. To examine the functional relevance of this interaction, fibroblasts were transfected with caveolin-1 siRNA and found to accumulate increased amounts of LDL-derived cholesterol within late endosomes/ lysosomes. Together, this report presents novel results demonstrating that the NPC1 and caveolin-1 proteins interact to modulate efflux of LDL-derived cholesterol from late endocytic compartments.

The human Niemann-Pick C1 (NPC1) gene has been found to be associated with extreme (early-onset and morbid-adult) obesity and type 2 diabetes independent of body weight. We previously performed growth studies using BALB/cJ Npc1 normal (Npc1+/+) and Npc1 heterozygous (Npc1+/-) mice and determined that decreased Npc1 gene dosage interacts with a high-fat diet to promote weight gain and adiposity. The present study was performed using both BALB/cJ and C57BL/6J Npc1+/+ and Npc1+/- mice to determine if decreased Npc1 gene dosage predisposes to metabolic features associated with type 2 diabetes. The results indicated that C57BL/6J Npc1+/- mice, but not BALB/cJ Npc1+/- mice, have impaired glucose tolerance when fed a low-fat diet and independent of body weight. The results also suggest that an accumulation of liver free fatty acids and hepatic lipotoxicity marked by an elevation in the amount of plasma alanine aminotransferase (ALT) may be responsible for hepatic insulin resistance and impaired glucose tolerance. Finally, the peroxisome-proliferator activated receptor α (PPARα) and sterol regulatory element-binding protein-1 (SREBP-1) pathways known to have a central role in regulating free fatty acid metabolism were downregulated in the livers, but not in the adipose or muscle, of C57BL/6J Npc1+/- mice compared to C57BL/6J Npc1+/+ mice. Therefore, decreased Npc1 gene dosage among two different mouse strains interacts with undefined modifying genes to manifest disparate yet often related metabolic diseases., (Published by Elsevier B.V.)

Objective: The goal of this study was to investigate the effects of a high-fat diet supplemented with fish oil or olive oil, fed to C57BL/6J mice for an extended period, on metabolic features associated with type 2 diabetes., Methods: Mice were fed one of four diets for 30 wk: a low-fat diet, a high-fat diet supplemented with lard, a high-fat diet supplemented with fish oil, or a high-fat diet supplemented with olive oil. Phenotypic and metabolic analysis were determined at 15 and 25 to 30 wk, thereby providing comparative analysis for weight gain, energy consumption, fat distribution, glucose and insulin tolerance, and hepatic/plasma lipid analysis., Results: Mice fed a high-fat diet supplemented with fish oil had improved glucose tolerance after an extended period compared with mice fed a high-fat diet supplemented with lard. Moreover, mice fed a high-fat diet supplemented with fish oil had significantly decreased concentrations of liver cholesterol, cholesteryl ester, and triacylglycerol compared with mice fed a high-fat diet supplemented with either lard or olive oil., Conclusion: Mice fed a high-fat diet supplemented with fish oil improved metabolic features associated with type 2 diabetes such as impaired glucose tolerance and hepatic steatosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The genes contributing to childhood obesity are categorized into three different types based on distinct genetic and phenotypic characteristics. These types of childhood obesity are represented by rare monogenic forms of syndromic or non-syndromic childhood obesity, and common polygenic childhood obesity. In some cases, genetic susceptibility to these forms of childhood obesity may result from different variations of the same gene. Although the prevalence for rare monogenic forms of childhood obesity has not increased in recent times, the prevalence of common childhood obesity has increased in the United States and developing countries throughout the world during the past few decades. A number of recent genome-wide association studies and mouse model studies have established the identification of susceptibility genes contributing to common childhood obesity. Accumulating evidence suggests that this type of childhood obesity represents a complex metabolic disease resulting from an interaction with environmental factors, including dietary macronutrients. The objective of this article is to provide a review on the origins, mechanisms, and health consequences of obesity susceptibility genes and interaction with dietary macronutrients that predispose to childhood obesity. It is proposed that increased knowledge of these obesity susceptibility genes and interaction with dietary macronutrients will provide valuable insight for individual, family, and community preventative lifestyle intervention, and eventually targeted nutritional and medicinal therapies.

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over-accumulation of low-density lipoprotein-derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three-hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP-1 via its acidic/di-leucine motif. Consequently, a nonfunctional AP-1A cytosolic complex resulted in a typical NPC-like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ-cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP-1A is essential for the lysosomal targeting and function of NPC1 and NPC2., (© 2013 John Wiley & Sons A/S.)

The Niemann-Pick C1 (NPC1) gene is associated with human obesity. Mouse models with decreased Npc1 gene dosage are susceptible to weight gain when fed a high-fat diet, but not a low-fat diet, consistent with an Npc1 gene-diet interaction. The objectives of this study were to define regulation of the Npc1 gene and to investigate the Npc1 gene-diet interaction responsible for weight gain. The experimental design involved feeding C57BL/6J male mice a low-fat diet (with 0.00, 0.10, or 1.00% cholesterol) or a high-fat diet (with 0.02% cholesterol) until 30 wk to determine regulation of the Npc1 gene in liver. The key results showed that the Npc1 gene was downregulated by dietary fatty acids (54%, P = 0.022), but not by dietary cholesterol, through feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway. However, the dietary fatty acids secondarily increased liver cholesterol, which also inhibits the SREBP pathway. Similarly, the Npc1 gene was downregulated in peritoneal fibroblasts isolated from C57BL/6J weanling male mice not exposed to the experimental diets and incubated in media supplemented with purified oleic acid (37%, P = 0.038) but not in media supplemented with purified cholesterol. These results are important because they suggest a novel mechanism for the interaction of fatty acids with the Npc1 gene to influence energy balance and to promote weight gain. Moreover, the responsiveness of the Npc1 gene to fatty acids is consistent with studies that suggest that the encoded NPC1 protein has a physiologic role in regulating both cholesterol and fatty acid metabolism.

Childhood overweight and obesity have reached epidemic proportions worldwide, and the increase in weight-associated co-morbidities including premature type 2 diabetes mellitus (T2DM) and atherosclerotic cardiovascular disease will soon become major healthcare and economic problems. A number of studies now indicate that the childhood obesity epidemic which has emerged during the past 30 years is a complex multi-factorial disease resulting from interaction of susceptibility genes with an obesogenic environment. This review will focus on gene-diet interactions suspected of having a prominent role in promoting childhood obesity. In particular, the specific genes that will be presented (FTO, MC4R, and NPC1) have recently been associated with childhood obesity through a genome-wide association study (GWAS) and were shown to interact with nutritional components to increase weight gain. Although a fourth gene (APOA2) has not yet been associated with childhood obesity, this review will also present information on what now represents the best characterized gene-diet interaction in promoting weight gain.

A recent population-based genome-wide association study has revealed that the Niemann-Pick C1 (NPC1) gene is associated with early-onset and morbid adult obesity. Concurrently, our candidate gene-based mouse growth study performed using the BALB/cJ NPC1 mouse model (Npc1) with decreased Npc1 gene dosage independently supported these results by suggesting an Npc1 gene-diet interaction in relation to early-onset weight gain. To further investigate the Npc1 gene in relation to weight gain and metabolic features associated with insulin resistance, we interbred BALB/cJ Npc1(+/-) mice with wild-type C57BL/6J mice, the latter mouse strain commonly used to study aspects of diet-induced obesity and insulin resistance. This breeding produced a hybrid (BALB/cJ-C57BL/6J) Npc1(+/-) mouse model with increased susceptibility to weight gain and insulin resistance. The results from our study indicated that these Npc1(+/-) mice were susceptible to increased weight gain characterized by increased whole body and abdominal adiposity, adipocyte hypertrophy and hepatic steatosis in the absence of hyperphagia. Moreover, these Npc1(+/-) mice developed abnormal metabolic features characterized by impaired fasting glucose, glucose intolerance, hyperinsulinemia, hyperleptinemia and dyslipidemia marked by an increased concentration of cholesterol and triacylglycerol associated with low-density lipoprotein and high-density lipoprotein. The overall results are consistent with a unique Npc1 gene-diet interaction that promotes both weight gain and metabolic features associated with insulin resistance. Therefore, the NPC1 gene now represents a previously unrecognized gene involved in maintaining energy and metabolic homeostasis that will contribute to our understanding concerning the current global epidemic of obesity and type 2 diabetes mellitus.

A recent genome-wide association study has determined that the Niemann-Pick C1 (NPC1) gene is associated with early-onset and morbid adult obesity. However, what effects of the nonsynonymous variation in NPC1 on protein function result in weight gain remains unknown. The NPC1 heterozygous mouse model (Npc1(+/-)), which expresses one-half the normal amounts of functional Npc1 protein compared to the homozygous normal (Npc1(+/+)) mouse, was used to determine whether decreased Npc1 gene dosage was associated with weight gain when fed either a low-fat (10% kcal fat) or high-fat (45% kcal fat) diet beginning at 4 weeks of age until 20 weeks of age. The results indicated that Npc1(+/-) mice had significantly increased weight gain beginning at 13 weeks of age when fed a high-fat diet, but not when fed a low-fat diet, compared to the Npc1(+/+) mice fed the same diet. With respect to mice fed a high-fat diet, the Npc1(+/-) mice continued to have significantly increased weight gain to 30 weeks of age. At this age, the Npc1(+/-) mice were found to have increased liver and inguinal adipose weights compared to the Npc1(+/+) mice. Therefore, decreased Npc1 gene dosage resulting in decreased Npc1 protein function, promoted weight gain in mice fed a high-fat diet consistent with a gene-diet interaction.

Niemann-Pick type C1 disease (NPC1) is an autosomal recessive lysosomal storage disorder characterized by neonatal jaundice, hepatosplenomegaly, and progressive neurodegeneration. The present study provides the lipid profiles, mutations, and corresponding associations with the biochemical phenotype obtained from NPC1 patients who participated in the National NPC1 Disease Database. Lipid profiles were obtained from 34 patients (39%) in the survey and demonstrated significantly reduced plasma LDL cholesterol (LDL-C) and increased plasma triglycerides in the majority of patients. Reduced plasma HDL cholesterol (HDL-C) was the most consistent lipoprotein abnormality found in male and female NPC1 patients across age groups and occurred independent of changes in plasma triglycerides. A subset of 19 patients for whom the biochemical severity of known NPC1 mutations could be correlated with their lipid profile showed a strong inverse correlation between plasma HDL-C and severity of the biochemical phenotype. Gene mutations were available for 52 patients (59%) in the survey, including 52 different mutations and five novel mutations (Y628C, P887L, I923V, A1151T, and 3741&#95;3744delACTC). Together, these findings provide novel information regarding the plasma lipoprotein changes and mutations in NPC1 disease, and suggest plasma HDL-C represents a potential biomarker of NPC1 disease severity.

The Niemann-Pick C1 and C2 (NPC1 and NPC2) proteins have a central role in regulating the transport of lipoprotein-derived cholesterol from endocytic compartments to the endoplasmic reticulum for esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway. Since the NPC1 gene/protein has recently been shown to be downregulated by feedback inhibition of the SREBP pathway, the present study was performed to determine whether physiological downregulation of the NPC1 gene/protein alters the transport and metabolism of low-density lipoprotein (LDL)-derived cholesterol in human fibroblasts. To perform this study, three different culture conditions were used that included fibroblasts grown in lipoprotein-deficient serum (LPDS), LPDS supplemented with LDL, and LPDS supplemented with LDL, followed by equilibration in the absence of LDL to allow the transport of LDL-derived cholesterol from endocytic compartments and equilibration of cellular sterol pools. The results from this study indicated that in addition to the NPC1 gene/protein, the NPC2 gene/protein was also downregulated by LDL-derived cholesterol-dependent feedback inhibition and that downregulation of both the NPC1 and NPC2 genes/proteins was associated with the sequestration of LDL-derived cholesterol within endocytic compartments, including late endosomes/lysosomes after equilibration. Therefore, it is proposed that physiological and coordinate downregulation of the NPC1 and NPC2 genes/proteins promotes the sequestration of LDL-derived cholesterol within endocytic compartments and serves a role in maintaining intracellular cholesterol homeostasis.

The Niemann-Pick C1 (NPC1) protein regulates the transport of cholesterol from late endosomes/lysosomes to other compartments responsible for maintaining intracellular cholesterol homeostasis. The present study examined the expression of the NPC1 gene and the distribution of the NPC1 protein that resulted from the transport of LDL-derived cholesterol through normal human fibroblasts. A key finding was that the transport of cholesterol from late endosomes/lysosomes to the sterol-regulatory pool at the endoplasmic reticulum, as determined by feedback inhibition of the sterol-regulatory element binding protein (SREBP) pathway, was associated with the downregulation of the NPC1 gene. Consistent with these results, fibroblasts incubated with LDL had decreased amounts of SREBP protein that interacted with sterol-regulatory element (SRE) sequences positioned within the NPC1 gene promoter region. Finally, partial colocalization of the NPC1 protein with late endosomes/lysosomes and distinct regions of the endoplasmic reticulum suggested that the NPC1 protein may facilitate the transport of cholesterol directly between these two compartments. Together, these results indicate that the transport of LDL-derived cholesterol from late endosomes/lysosomes to the sterol-regulatory pool, known to be regulated by the NPC1 protein, is responsible for promoting feedback inhibition of the SREBP pathway and downregulation of the NPC1 gene.

Niemann-Pick type C1 (NPC1) disease is an autosomal recessive disorder characterized clinically by neonatal jaundice, hepatosplenomegaly, vertical gaze palsy, ataxia, dystonia, and progressive neurodegeneration. The present study provides basic clinical and health information from the National Niemann-Pick C1 disease database that was obtained using a clinical questionnaire of 83 questions mailed to families affected by NPC1 disease living in the United States. The study was conducted over a 1-year period, during which time parents/caregivers and physicians completed the clinical questionnaire. Sixty-four percent (87/136) of the questionnaires were returned, with 53% and 47% representing male and female NPC1 patients, respectively. The average age of diagnosis for NPC1 disease was 10.4 years, with one-half of patients being diagnosed before the age of 6.9 years. The average age of death for NPC1 disease was 16.2 years, with one-half of patients dying before the age of 12.5 years. A common clinical symptom reported at birth was neonatal jaundice (52%), followed by enlargement of the spleen (36%) and liver (31%); ascites (19%) and neonatal hypotonia (6%) were much less frequent. With respect to developmental difficulties, the most common findings included clumsiness (87%), learning difficulties (87%), ataxia (83%), dysphagia (80%), and vertical gaze palsy (81%). Together, these findings confirm and extend previous reports investigating the clinical features associated with NPC1 disease., (Copyright (c) 2007 Wiley-Liss, Inc.)

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.

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.

Niemann-Pick type C1 (NPC1) disease is a fatal hereditary disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. Although the NPC1 gene has been identified, the molecular mechanism responsible for neuronal dysfunction in brains of patients with NPC1 disease remains unknown. This study demonstrates that the amount of cholesterol within mitochondria membranes is significantly elevated in NPC1 mouse brains and neural cells. In addition, the mitochondrial membrane potential, the activity of ATP synthase, and henceforth the level of ATP are markedly decreased in NPC1 mouse brains and neurons. Importantly, reducing the level of cholesterol within mitochondrial membranes using methyl-beta-cyclodextrin can restore the activity of ATP synthase. Finally, NPC1 neurons show an impaired neurite outgrowth, which can be rescued by exogenous ATP. These results suggest that mitochondrial dysfunctions and subsequent ATP deficiency, which are induced by altered cholesterol metabolism in mitochondria, may be responsible for neuronal impairment in NPC1 disease.

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.

To maintain proper cellular function, the amount and distribution of cholesterol residing within cellular membranes must be regulated. The principal disorder affecting transport of cholesterol through the late endosomal/lysosomal system and intracellular cholesterol homeostasis is Niemann-Pick type C (NPC) disease. The genes responsible for NPC disease have been identified, and the encoded Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (HE1/NPC2) proteins are currently the subject of intense investigation. This review provides a detailed examination of NPC1 and HE1/NPC2 in regulating the transport of cholesterol through the late endosomal/lysosomal system to other cellular compartments responsible for maintaining intracellular cholesterol homeostasis, and how defective function of these proteins may be responsible for the pathophysiology associated with NPC disease.

Niemann-Pick type C (NPC) disease is a rare inherited metabolic disorder characterized by hepatosplenomegaly, progressive neurodegeneration, and storage of lipids such as cholesterol and glycosphingolipids in most tissues. The current study was conducted to characterize the Niemann-Pick C1 (NPC1) protein in feline fibroblasts. This was accomplished by generating rabbit polyclonal antibodies against a peptide corresponding to amino acids 1256-1275 of the feline NPC1 protein. The results obtained using immunoblot analysis identified two major proteins that migrated at approximately 140 and 180 kDa. These two proteins were absent when immunoblots were incubated in the presence of feline NPC1 antibody and immunizing peptide, or preimmune serum. Fluorescence microscopy of feline fibroblasts incubated with the feline NPC1 antibody revealed granular staining within the perinuclear region of the cell. This granular staining was diminished when feline fibroblasts were incubated in the presence of feline NPC1 antibody and immunizing peptide, or was completely absent when feline fibroblasts were incubated in the presence of preimmune serum. Additional studies using double-labeled fluorescence microscopy indicated that feline NPC1 partially colocalized with markers for late endosomes/lysosomes, endoplasmic reticulum, and microtubules, but not the trans-Golgi network. In summary, the results presented in this report demonstrate that the NPC1 protein in feline fibroblasts has a similar distribution as that previously described for human and murine fibroblasts.

The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar. These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae.

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.

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., (Copyright 2001 Academic Press.)

Niemann-Pick disease type C (NPC) is characterized by an accumulation of unesterified cholesterol in the endosomal/lysosomal (E/L) system, resulting in progressive neurodegeneration and death during early childhood. To investigate the cellular pathomechanism of nervous system involvement in NPC, continuous neural cell lines are desirable. In this study, we obtained neuronal and Schwann cell cultures and established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of NPC model mouse (spm/spm). One of the cell lines, designated SPMS9, had distinct Schwann cell phenotypes and was maintained over 10 months without phenotypic alterations. The level of Npc1 mRNA was markedly decreased, and NPC1 protein was not detectable in SPMS9 cells. These cells contained intracytoplasmic granules positive for filipin cholesterol staining and immunoreactive for GM2 ganglioside. Electron-microscopically, intracytoplasmic polymorphous membranous inclusions and vacuoles were demonstrated in SPMS9 cells. The treatment with an inhibitor of ceramide-specific glucosyltransferase, N-butyldeoxynojirimysin (NB-DNJ) markedly reduced the intracytoplasmic granular immunofluorescence for GM2 ganglioside in SPMS9 cells, whereas the amount of filipin-positive granules remained unchanged. The SPMS9 cells retained vesicular fluorescence of cationic dye acriflavine 16-24 hours after loading, indicating the defect of transmembrane efflux pump activity of NPC1 in the E/L compartment in these cells. These immortalized Schwann cells can be useful in studies on the nervous system lesions in NPC.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

Mouse Niemann-Pick disease type C1 (npc1), formerly designated spm (sphingomyelinosis), is an autosomal recessive lipid storage disorder. We generated a high-resolution linkage map in the 2.24-cM npc1 critical region by typing eight polymorphic markers in 2322 meioses (948 of these were previously reported). A minimal set of overlapping yeast artificial chromosomes (YACs) had previously been assembled (Hsu and Erickson 2000). The YAC 313-B-8, which covered this whole region, has been used to construct cosmid libraries. Three cosmid contigs were built, and one of them contained the npc1 locus. Two (CA)(n) microsatellites were identified, and the one new one was characterized, from the YAC-derived cosmids. The most proximal cosmid contig overlaps with markers near twirler (Tw). Both the physical map and genetic linkage map have been integrated to study the recombination frequencies in this particular region of the mouse genome, and recombination suppression due to the heterozygous insertion of DNA was suggested.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.

Niemann-Pick type C (NPC) is a neurodegenerative disorder with somatically altered cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequences shared with known sterol-sensing proteins. We have used a mouse model with a disrupted Npc1 gene to study two cholesterol-lowering drugs (nifedipine and probucol) and the effects of introducing a null mutation in the low-density lipoprotein receptor (LDLR). Although these treatments significantly ameliorated liver cholesterol storage, little effect on the onset of neurological symptoms was found.

This study assesses the developmental expression of the Niemann-Pick type C mRNA in vivo and in vitro in rat cerebellum. NPC is an autosomal recessive neurovisceral lipid storage disease associated with an alteration in cholesterol trafficking. In the mouse model of NPC and in the early onset form of human NPC, Purkinje neurons are among the first neurological targets, suffering stunted growth during postnatal development and dying, leading to ataxia. Recently, the genes responsible for human (NPC1) and mouse (Npc1) NPC disease have been cloned. Based on a highly homologous domain, we designed primers to look for levels of Npc1 mRNA with a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) approach using cyclophilin as an internal standard. Total RNA was isolated from various postnatal developmental stages of the rat cerebellum as template for the analyses. Npc1 transcripts were observed at postnatal day 0 and at later stages of development, both in vivo and in vitro from primary cerebellar cultures. To identify the location of Npc1 inside the cerebellum, we performed immunostaining with an anti-Npc1 antibody in primary rat cerebellar cultures identifying reactive Purkinje neurons by double-labeling with the Purkinje specific marker calbindin and sub-populations of glial cells. In summary, Npc1 is expressed in rat cerebellum in vivo and in vitro and is expressed during early postnatal development as well as in the adult cerebellum. Since Npc1 is expressed at similar levels throughout development, the vulnerability of Purkinje neurons to this disease is likely to involve disruption of an interaction with other developmentally-regulated proteins.

We have previously demonstrated (1) an increased expression of caveolin-1 in murine heterozygous and homozygous Niemann-Pick type C (NPC) livers, and (2) an increased concentration of unesterified cholesterol in a detergent insoluble caveolae-enriched fraction from homozygous livers. To define further the relationship between caveolin-1 function and the cholesterol trafficking defect in NPC, we examined the expression and distribution of additional caveolar and signal transduction proteins. The expression of annexin II was significantly increased in homozygous liver homogenates and the Triton X-100 insoluble floating fraction (TIFF). Phosphoamino acid analysis of caveolin-1 and annexin II from the homozygous TIFF demonstrated an increase in serine and tyrosine phosphorylation, respectively. To determine the basis for increased phosphorylation of these proteins, the expression and distribution of several protein kinases was examined. The expression of PKCalpha, PKCzeta and pp60-src (protein kinases) were significantly increased in both heterozygous and homozygous liver homogenates, while PKCdelta was increased only in homozygous livers. Of the protein kinases analyzed, only CK IIalpha was significantly enriched in the heterozygous TIFF. Finally, the concentration of diacylglycerol in the homozygous TIFF was significantly increased and this elevation may modulate PKC distribution and function. These results provide additional evidence for involvement of a caveolin-1 containing cellular fraction in the pathophysiology of NPC and also suggest that the Npc1 gene product may directly or indirectly, regulate the expression and distribution of signaling molecules.

Interaction of HDL with cells activates protein kinase C (PKC), a process that may be important in stimulating efflux of excess cellular cholesterol. Here we report that HDL treatment of cholesterol-loaded fibroblasts increases 32P labeling of three acidic phosphoproteins. These phosphoproteins, called pp80, pp27, and pp18 based on apparent M(r) in kD, were also phosphorylated by acute treatment of cells with phorbol myristate acetate, suggesting that they are regulated in response to PKC activation. The HDL-stimulated phosphorylation of pp80 and pp18 was significant after only 30 seconds and was sustained for at least 30 and 120 minutes, respectively, while increased phosphorylation of pp27 was transient, reaching a maximum at 10 minutes. Both pp27 and pp18 were phosphorylated on serine/threonine residues, whereas pp80 was phosphorylated on serine/threonine and tyrosine residues. Immunoprecipitation studies suggested that pp80 is the myristoylated alanine-rich C kinase substrate protein, but the identities of pp27 and pp18 are unknown. HDL and trypsin-digested HDL stimulated phosphorylation of pp80 and pp27, while purified apoA-I, apoA-II, or apoE had no stimulatory effects, indicating that the active component in HDL was trypsin resistant and unlikely to be an apolipoprotein. Conversely, HDL, apoA-I, apoA-II, and apoE all stimulated pp18 phosphorylation, while trypsin-digested HDL had less effect, consistent with pp18's being responsive to HDL apolipoproteins. Treatment of cholesterol-depleted cells with apoA-I also stimulated phosphorylation of pp18, but only transiently. These results suggest that HDL interaction with cells activates diverse PKC-mediated pathways that target different phosphoproteins. Of these three phosphoproteins, only pp18 has a phosphorylation response consistent with its being involved in apolipoprotein-mediated lipid transport.

Niemann-Pick type C (NPC) is an autosomal recessive disease characterized by impaired cholesterol homeostasis due to a defect in the intracellular transport of unesterified cholesterol. While accumulation of lysosomal cholesterol is the most apparent microscopic finding, cholesterol has also been shown to accumulate in the trans-cisternae of the Golgi apparatus. Caveolin-1, a cholesterol-binding protein that cycles between the Golgi apparatus and the plasma membrane, has been hypothesized to participate in cholesterol transport. Using the BALB/c murine model for NPC disease, we found that the expression of caveolin-1 in total liver homogenates from heterozygous and homozygous affected animals was altered. Immunoblot analysis of liver homogenates from heterozygous mice revealed that caveolin-1 is significantly (p < 0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from homozygous affected mice also had a significant (p < 0.05) increase in caveolin-1 expression, 1.6 fold, compared to normal mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin-1 was localized to sinusoidal endothelial cells in heterozygous mice. The Triton insoluble floating fraction (TIFF) was isolated using liver from each genotype and analyzed for caveolin-1 expression. Caveolin-1 in the TIFF from heterozygous mice was significantly (p < 0.01) elevated, 1.8 fold, compared to normal and homozygous affected animals; normal and homozygous affected animals, however, were not significantly different from each other. The TIFF isolated from homozygous affected mice revealed a 15 fold increase in unesterified cholesterol compared to the TIFF isolated from heterozygous and normal mice. In summary, these findings demonstrate an altered expression of caveolin-1 in liver from heterozygous and homozygous NPC mice and increased concentration of cholesterol from TIFF in homozygous affected NPC mice. The identification of these alterations in the TIFF suggests involvement of detergent insoluble membrane structures, possibly caveolae and/or detergent insoluble glycosphingolipid-enriched complexes (DIGs), in the cholesterol trafficking defect in this disorder.

Human Niemann-Pick type II fibroblasts, which encompass the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) variants, exhibit altered expression of caveolin-1 protein when examined by immunoblotting using an anti-caveolin-1 monoclonal antibody. Unexpectedly, caveolin-1 in heterozygous fibroblasts was significantly elevated as much as 10-fold compared to caveolin-1 in normal and homozygous affected fibroblasts. Homozygous NPC fibroblasts expressed caveolin-1 levels similar to those in normal fibroblasts, while the expression of caveolin-1 in homozygous NPD fibroblasts was slightly elevated. Northern analysis indicates that normal fibroblasts and NPC heterozygous fibroblasts have similar amounts of caveolin-1 mRNA, while NPC homozygous fibroblasts have significantly less caveolin-1 mRNA. In contrast, heterozygous and homozygous NPD fibroblasts exhibit increased levels of caveolin-1 mRNA. These novel findings suggest that caveolin-1 containing subcellular structures are involved in the pathophysiology of Niemann-Pick type II disease. Furthermore, altered caveolin-1 protein expression may serve as a useful marker for the diagnosis of carriers of NPC or NPD.

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.