Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by endolysosomal cholesterol accumulation. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that non-enzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1−/− mouse model and found several cholesterol oxidation products that were elevated in Npc1−/− mice, were detectable prior to the onset of symptoms, and were associated with disease progression. Non-enzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

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Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1 I1061T , is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1 I1061T mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1 I1061T mutation. Despite similar NPC1 mRNA levels between wild type and NPC1I1061T fibroblasts, NPC1 protein levels are decreased by 85% in NPC1 I1061T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1I1061T protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t1 ⁄ 2 6.5 h) versus wild type Endo H-resistant species (t1 ⁄ 2 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1I1061T protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1 I1061T in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1 I1061T protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1 I1061T mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1 I1061T mutation.

Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eighttransmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of Zinc is an essential trace element that is required for the catalytic activity of numerous metalloenzymes (4, 5) and can also serve a purely structural role by stabilizing the conformation of certain zinc-dependent protein domains, such as zinc fingers, zinc clusters, and RING fingers, that are commonly found in transcriptional regulatory proteins (5, 6). Deficiency of this essential metal can have devastating effects. In mammals, inadequate levels of zinc in the diet lead to dermatologic lesions, growth retardation, mental disorders, and compromised function of the immune and reproductive systems (7-9). Likewise, high levels of zinc can be cytotoxic. Thus, cells must maintain tight control over intracellular zinc levels. This control is achieved through a balance of zinc efflux, cellular zinc storage, and zinc uptake. Each of these activities is mediated by a distinct family of proteins (8).In mammals, the zinc transporter (ZnT) 1 family of proteins function in a tissue-, cell-, and organelle-specific manner to regulate intracellular zinc homeostasis. These proteins contain six predicted transmembrane domains and are thought to function as multimers. Seven members of the Znt family have been identified to date in mammals (Znt1-7), and genetic studies have confirmed the importance of many of these genes in mammalian zinc metabolism (10 -17). Recent studies demonstrated that zinc can regulate expression of the Znt1 gene (18), and dietary zinc modulates Znt1 and Znt2 mRNA levels in the kidney, intestine, and liver (19). Zinc can also regulate the intracellular localization of the ZnT4 and ZnT6 proteins (16).Intracellular zinc is bound by small cysteine-rich proteins called metallothioneins (MTs) (20). In the mouse, the MT family consists of four members. Exposure to high levels of zinc causes MT accumulation through increased gene expression, whereas dietary zinc deficiency leads to decreased MT abundance through decreased gene expression and protein destabilization (21). These proteins are thought to sequester zinc when present at high levels, protecting against heavy metal toxicity, and to provide a labile pool of zinc under limiting conditions that can be released for use by other proteins.In eukaryotes, uptake of several essential metals is mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters (22, 23). The first member of this family to be i...

Niemann-Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1 ؊/؊ mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1 ؊/؊ mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1 ؊/؊ mice involves GABAA receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABA A receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1 ؊/؊ mice, strongly supporting a GABAA receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease.cholesterol ͉ neurosteroid ͉ allopregnanolone ͉ neurodegeneration N iemann-Pick type C (NPC) disease is an autosomal recessive neurodegenerative disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system and patterned Purkinje cell death in the cerebellum (1). Mutations in the NPC1 gene are responsible for Ϸ95% of human NPC disease. NPC1 loss-of-function mutants exhibit marked impairment of low-density lipoprotein (LDL) cholesterol esterification and mobilization of newly hydrolyzed LDL cholesterol to the plasma membrane (2-4), resulting in lysosomal sequestration of LDL cholesterol, delayed down-regulation of the LDL receptor and de novo cholesterol biosynthesis, and impaired ABCA1-mediated cholesterol efflux (5-7). Despite recent progress in characterizing the biochemical and genetic defects in NPC disease, the mechanisms underlying the neurodegenerative phenotype are not well understood. Moreover, at present there are no effective therapies that delay progression of human NPC disease.Many of the prominent neuropathological features of human NPC disease [e.g., neuronal lipid storage and progressive loss of Purkinje neurons (1)] are recapitulated in the BALB͞c NPC nih (npc1 Ϫ/Ϫ ) mouse, a naturally occurring murine model that harbors a retroposon insertion in the Npc1 gene (8, 9). In NPC1 mice, accumulation of unesterified cholesterol and gangliosides occurs in morphologically normal neurons as early as postnatal day 9 (P9) and precedes neuronal injury and c...

Niemann-Pick C1 (NPC1) is a key participant in cellular cholesterol trafficking. Loss of NPC1 function leads to defective suppression of SREBP-dependent gene expression and failure to appropriately activate liver X receptor-mediated (LXR-mediated) pathways, ultimately resulting in intracellular cholesterol accumulation. To determine whether NPC1 contributes to regulation of macrophage sterol homeostasis in vivo, we examined the effect of NPC1 deletion in BM-derived cells on atherosclerotic lesion development in the Ldlr -/-mouse model of atherosclerosis. High-fat diet-fed chimeric Npc1 -/-mice reconstituted with Ldlr -/-Npc1 -/-macrophages exhibited accelerated atherosclerosis despite lower serum cholesterol compared with mice reconstituted with wild-type macrophages. The discordance between the low serum lipoprotein levels and the presence of aortic atherosclerosis suggested that intrinsic alterations in macrophage sterol metabolism in the chimeric Npc1 -/-mice played a greater role in atherosclerotic lesion formation than did serum lipoprotein levels. Macrophages from chimeric Npc1 -/-mice showed decreased synthesis of 27-hydroxycholesterol (27-HC), an endogenous LXR ligand; decreased expression of LXR-regulated cholesterol transporters; and impaired cholesterol efflux. Lower 27-HC levels were associated with elevated cholesterol oxidation products in macrophages and plasma of chimeric Npc1 -/-mice and with increased oxidative stress. Our results demonstrate that NPC1 serves an atheroprotective role in mice through regulation of LXR-dependent cholesterol efflux and mitigation of cholesterol-induced oxidative stress in macrophages. IntroductionMacrophages regulate tissue lipid homeostasis through the uptake and catabolism of atherogenic plasma lipoproteins (1). Macrophages express scavenger receptor A and CD36, which bind and internalize modified lipoproteins, such as oxidized LDL. Upon internalization, lipoprotein-derived cholesterol is delivered to a late endosomal organelle, where cholesterol esters are hydrolyzed to free cholesterol. Delivery of free cholesterol from the endocytic pathway to the plasma membrane and ER requires the coordinated actions of the late endosomal Niemann-Pick C1 (NPC1) and lysosomal NPC2 proteins (2). Delivery of cholesterol to the ER rapidly stimulates esterification and accumulation of cholesterol ester in cytoplasmic droplets. Because the scavenger receptors, in contrast to the LDL receptor (LDLR), are not controlled by a sterol-regulated negative feedback loop, macrophages accumulate massive quantities of lipoprotein-derived lipids.

Background:The mechanisms regulating internalization of plasma membrane cholesterol in mammalian cells are not well understood. Results: A cell line haploinsufficient for U60 snoRNA expression exhibits impaired plasma membrane to ER cholesterol trafficking and increased de novo cholesterol synthesis. Conclusion: U60 snoRNA expression regulates cholesterol homeostasis by affecting internalization of plasma membrane cholesterol. Significance: This is the first study to implicate a snoRNA in regulation of cholesterol homeostasis.

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