A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.     

Do mammalian NPC1 and NPC2 play a role in intestinal cholesterol absorption?

NPC1L1 (Niemann-Pick C1-like 1), the pharmacological target of the cholesterol-uptake inhibitor ezetimibe, is a transporter localized on the brush border of enterocytes. Although this protein plays a key role in intestinal uptake of sterols, multiple molecular events that underlie intestinal cholesterol absorption have not been fully characterized. Two proteins that might be involved in this process are NPC1 and NPC2 (Niemann-Pick disease type C proteins 1 and 2), which function in the endosomal/lysosomal cholesterol egress pathway and whose deficiency results in NPC (Niemann-Pick type C) disease. The involvement of these proteins in intestinal cholesterol absorption was examined in mutant mice lacking either NPC1 or NPC2. Our data indicate that deficiencies in either protein do not have an effect on cholesterol uptake or absorption. This contrasts with recent results obtained for the fruitfly Drosophila melanogaster, which indicate that a deficiency of NPC1 (dNPC1a being its Drosophila homologue) leads to activation of an NPC1L1 (Drosophila homologue dNPC1b)-independent cholesterol uptake pathway, underscoring fundamental differences in mammalian and non-mammalian cholesterol metabolism.     

Drosophila NPC1b promotes an early step in sterol absorption from the midgut epithelium

The NPC1 family of proteins plays crucial roles in the intestinal absorption and intracellular trafficking of sterols. The Drosophila genome encodes two NPC1 homologs, one of which, NPC1a, is required for intracellular sterol trafficking in many tissues. Here we show that the other Drosophila NPC1 family member, NPC1b, is expressed in the midgut epithelium and that NPC1b is essential for growth during the early larval stages of development. NPC1b mutants are severely defective in sterol absorption, and the midgut epithelium of NPC1b mutants is deficient in sterols and sterol trafficking intermediates. By contrast, NPC1a mutants absorb sterols more efficiently than wild-type animals, and, unexpectedly, NPC1b;NPC1a double mutants absorb sterols as efficiently as wild-type animals. Together, these findings suggest that NPC1b plays an early role in sterol absorption, although sterol absorption continues at high efficiency through an NPC1a- and NPC1b-independent mechanism under conditions of impaired intracellular sterol trafficking.     

Secretion of sterols and the NPC2 protein from primary astrocytes

Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.     

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

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

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time approximately 2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.     

Deficiency of a Niemann-Pick, type C1-related protein in toxoplasma is associated with multiple lipidoses and increased pathogenicity

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved 'sterol-sensing domain' (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.     

Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects.

In mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss-of-function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain-of-function allele, Tumorous-lethal (hopTum-l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum-l mutation. Overexpression of either wild-type hop or hopTum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum-l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum-l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum-l results in greater phosphorylation than overexpression of the wild-type. We conclude that hopTum-l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells.     

Sterols and intracellular vesicular trafficking: lessons from the study of NPC1

Cholesterol is an important structural component of membranes as well as a precursor for steroid hormone, bile acid and regulatory oxysterol biosynthesis. Recent observations revealed that cholesterol plays an important role in signaling and the regulation of intracellular vesicular trafficking. Studies on Niemann-Pick type C disease, a fatal neuro-visceral cholesterol storage disorder, led to the elucidation of a sterol-modulated vesicular trafficking pathway. Mutations in the NPC1 gene, which cause the majority of cases of Niemann-Pick type C disease, result in the accumulation of free cholesterol in lysosomes and associated defects in glycolipid sorting. NPC1 has a sterol-sensing domain that presumably recognizes free sterols in the protein's environment and participates in the movement of cholesterol out of lysosomes. The compartment containing NPC1 is a subset of late endosomes; it is highly mobile, travels along microtubules, emitting flexible tubules. The movements of this compartment require an intact NPC1 sterol-sensing domain and are dramatically suppressed when free cholesterol accumulates in the late endosomes. Two other proteins involved in sterol trafficking enter into the NPC1 compartment, NPC2 also known as HE1, a secreted sterol-binding glycoprotein, and MLN64, a StAR-related lipid transfer (START) domain protein, which can bind cholesterol and promote its movement from donor to acceptor membranes. Mutations in NPC2 cause a rarer form of Niemann-Pick type C disease, establishing its importance in intracellular sterol movement. NPC2, NPC1 and MLN64 may act in an ordered sequence to sense cholesterol, effect sterol movement, and consequently, influence the process of vesicular trafficking.     

The Niemann-Pick C1 gene is downregulated by feedback inhibition of the SREBP pathway in human fibroblasts

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.     

Genes expressed in the ring gland,the major endocrine organ of Drosophila melanogaster.

We have used an enhancer-trap approach to begin characterizing the function of the Drosophila endocrine system during larval development. Five hundred and ten different lethal PZ element insertions were screened to identify those in which a reporter gene within the P element showed strong expression in part or all of the ring gland, the major site of production and release of developmental hormones, and which had a mutant phenotype consistent with an endocrine defect. Nine strong candidate genes were identified in this screen, and eight of these are expressed in the lateral cells of the ring gland that produce ecdysteroid molting hormone (EC). We have confirmed that the genes detected by these enhancer traps are expressed in patterns similar to those detected by the reporter gene. Two of the genes encode proteins, protein kinase A and calmodulin, that have previously been implicated in the signaling pathway leading to EC synthesis and release in other insects. A third gene product, the translational elongation factor EF-1alpha F1, could play a role in the translational regulation of EC production. The screen also identified the genes couch potato and tramtrack, previously known from their roles in peripheral nervous system development, as being expressed in the ring gland. One enhancer trap revealed expression of the gene encoding the C subunit of vacuolar ATPase (V-ATPase) in the medial cells of the ring gland, which produce the juvenile hormone that controls progression through developmental stages. This could reveal a function of V-ATPase in the response of this part of the ring gland to adenotropic neuropeptides. However, the gene identified by this enhancer trap is ubiquitously expressed, suggesting that the enhancer trap is detecting only a subset of its control elements. The results show that the enhancer trap approach can be a productive way of exploring tissue-specific genetic functions in Drosophila.     

A model for niemann-pick type C disease in the nematode Caenorhabditis elegans

Niemann-Pick type C (NP-C) disease is a progressive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in lysosomes [1]. NP-C patients show various defects including hepatosplenomegaly, ataxia, dystonia and dementia. Most cases of NP-C are associated with inactivating mutations of the NPC1 gene [2], which encodes a protein implicated in the retrograde transport of sterols and other cargo from lysosomes [3]. Furthermore, localization of the NPC1 protein to lysosomal/endosomal compartments is essential for proper transport [4]. To create a model of NP-C disease in a simple, genetically tractable organism, we generated deletion mutations in two Caenorhabditis elegans homologs of the human NPC1 gene, designated npc-1 and npc-2. Animals mutant for npc-1 developed slowly, laid eggs prematurely, and were hypersensitive to cholesterol deprivation. Furthermore, npc-1; npc-2 double-mutant animals inappropriately formed dauer larvae under favorable growth conditions. These phenotypes in C. elegans provide a model system for both genetic and chemical suppressor screening that could identify promising drug targets and leads for NP-C disease.     

Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop

Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [(3)H]cholesterol (K(d), 130 nM) and [(3)H]25-hydroxycholesterol (25-HC, K(d), 10 nM) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [(3)H]cholesterol, but weakly for [(3)H]25-HC binding. Binding of [(3)H]cholesterol but not [(3)H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [(3)H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [(3)H]cholesterol and [(3)H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.     

Structure and function of the NPC2 protein

Somatic cell hydridization and linkage studies indicated the implication of a second gene as a cause of Niemann-Pick C disease in a minority (5%) of patients. A study of the lysosomal proteome led to the identification of a previously known gene, HE1, as the NPC2 gene. The mature NPC2/HE1 protein is a ubiquitous soluble small 132-amino-acid glycoprotein, first characterized as a major secretory protein in the human epididymis, but also detected in most tissues. Seventeen families with mutations in the NPC2 gene are known. Good genotype-phenotype correlations were observed. No distinction can be made between the biochemical phenotypes of NPC1 or NPC2 mutants. The NPC2 protein binds cholesterol with submicromolar affinity at neutral and acidic pH. The bovine protein has been crystallized, and the cholesterol-binding site assigned to a hydrophobic loosely packed region. There is strong evidence that the NPC1 and NPC2 proteins must function in a closely related fashion. Current data have led to the hypothesis that NPC2 would bind cholesterol from internal lysosomal membranes, enabling a physical interaction with NPC1 (or another protein) and allowing postlysosomal export of cholesterol. In this model, the activity of NPC1 would depend on that of NPC2. The precise function of the NPC2 protein has, however, not been fully elucidated.     

Lipid trafficking defects increase Beclin-1 and activate autophagy in Niemann-Pick type C disease

Niemann-Pick type C disease (NPC) is a sphingolipid storage disorder characterized by progressive neurodegeneration that typically shows juvenile onset. Mutations in the Npc1 gene cause approximately 95% of NPC cases. NPC1 is a multipass transmembrane protein involved in lipid and cholesterol trafficking. Loss of function mutations in Npc1 lead to the accumulation of sphingolipids and cholesterol in late endosomes and lysosomes. In our study, we demonstrated that NPC1 deficiency results in increased basal autophagy in human fibroblasts and in mice. We further demonstrated that NPC1 deficiency activate basal autophagy through increased expression of Beclin-1, a highly conserved member of the class III PI3K complex that is critical for the formation of autophagosomes. In contrast, enhanced basal autophagy was not associated with activation of the Akt-mTORp70 S6K signaling pathway. Increased Beclin-1 levels and elevated autophagy were also observed in other sphingolipid storage diseases characterized by disrupted cholesterol and sphingolipid trafficking. We propose a model in which the disordered cholesterol trafficking that occurs in many sphingolipid storages diseases results in upregulation of Beclin-1 and enhanced levels of autophagy.     

Cellular Cholesterol Storage in the Niemann-Pick Disease Type C Mouse Is Associated with Increased Expression and Defective Processing of Apolipoprotein D

Abstract: Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.