InteractiveFly: GeneBrief

Niemann-Pick Type C-1a : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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. A Drosophila NPC model has been created by mutating NPC1, referred to in this study as 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 develop only to the first larval stage and are 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 allows development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. It is proposed 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 (Huang, 2005; Fluegel, 2005).

Niemann-Pick Type C disease (NPC) is both a lysosomal storage disorder and a neurodegenerative disease. At the cellular level, the most notable aspect of the disease is a massive accumulation of cholesterol, glycosphingolipids and other lipids in aberrant organelles. The underlying defect appears to be a failure of normal organelle trafficking and a consequent failure of lipid homeostasis (Liscum, 2004; Mukherjee, 2004; Sturley, 2004). The disease is inherited as an autosomal recessive condition. The disease is caused by mutations in either of two genes, NPC1 (Carstea, 1997) and NPC2 (Naureckiene, 2000; Huang, 2005 and references therein).

Neurons in individuals with NPC gene mutations and in a cat model of the disease grow extra dendritic processes and form neurofibrillary tangles (NFTs) (Walkley, 2004), and progressive neurodegeneration is a prominent symptom, particularly in the cerebellum (Higashi, 1993). Options for therapy are highly limited at this time (Patterson, 2004). The outcome for individuals with NPC is usually death in the teenage years. Understanding the origins of NPC is important to find ways to save lives and because it provides an entry point for studying still-mysterious aspects of lipid and transport cell biology (Huang, 2005).

The two NPC genes encode entirely different types of proteins that probably participate in the same pathway, though the molecular mechanisms that link the two proteins are unknown (Vanier, 2003). NPC1, a cholesterol binding (Ohgami, 2004) 13 transmembrane region protein (Davies, 2000a and Ioannou, 2000) is required for normal movements of populations of late endosomes and for proper homeostatic regulation of sterol and other lipid levels. Further interest in the NPC1 protein derives from its striking sequence similarity to the Patched protein (Carstea, 1997), the receptor for Hedgehog signaling proteins that regulate many aspects of growth and cell fate determination during development. Another closely related protein, NPC1L1, is crucial for intestinal uptake of cholesterol (Altmann, 2004; Davies, 2005). NPC1 works in a mysterious partnership with NPC2, a lysosomal protein that can be secreted and that binds strongly to cholesterol (Naureckiene, 2000; Friedland, 2003; Ko, 2003). The central mysteries still remain: what are the molecular functions of NPC1 and 2, how does either one regulate organelle movements and molecular trafficking, and why does loss of either protein lead to neurodegeneration and other symptoms (Huang, 2005)?

The npc1 gene is well conserved through about a billion years of evolution, allowing studies with a variety of powerful experimental organisms (Higaki, 2004). Useful NPC1 models have been generated using yeast (Malathi, 2004) and worm (Sym, 2000), although no sterol trafficking defect has been reported in either model. By mutating one of the two NPC1-like fly genes, dnpc1a, a Drosophila model of NPC1 has been created that has a cholesterol accumulation defect similar to that of mammalian NPC mutants. dnpc1a mutants accumulate sterol in a punctate pattern in many tissues, implying a conserved role of NPC1 in cholesterol trafficking from fly to mammals (Huang, 2005).

dnpc1a function is found to be crucial for normal steroid hormone metabolism. Neurosteroid treatment has been shown to suppress neurodegeneration in Npc1 mutant mice (Griffin, 2004), implying a neurosteroid hormone deficiency in Npc1 mice that may parallel the defect in Drosophila. Studies with dnpc1a mutants suggest a model for NPC1 function: the protein may allow delivery of sufficient sterol to mitochondria in order for steroid hormones to be made there. More complete understanding of the molecular and cell biology of these pathways may increase the options for NPC therapy (Huang, 2005).

Mutations in NPC1 cause large amounts of cholesterol and other lipids to accumulate in aberrant organelles in most, if not all, cells. The consequence for humans is typically, and tragically, neurodegeneration and other pathology leading to death in the teenage years. However, it is not clear to what extent sterol accumulation is the cause of pathology as opposed to an indicator of an intracellular trafficking process gone awry. The Drosophila model of NPC disease makes a strong case for the connection between NPC1 function and steroid production, reinforcing earlier studies with mice that had suggested a connection between NPC disease and neurosteroid production. The fly model offers the opportunity to study tissue-specific functions of Npc genes, and holds the promise of identifying interacting genes and proteins (Huang, 2005).

Although NPC1 is conserved through evolution, NPC1 mutants display specific and distinct phenotypes in different model organisms. From studies of worm, fly, and mouse NPC1 mutants, a possible evolutionarily conserved NPC1 function emerges: the protein is needed for cholesterol-derived hormone production. Owing to the diverse roles of steroid hormones in different organisms, NPC1 mutants have quite different phenotypes. The nematode C. elegans has two npc1-like genes: ncr-1 and ncr-2. Double ncr-1;ncr-2 mutants have a dauer constitutive phenotype. The dauer state is probably due to the shortage of a sterol-derived dauer formation-inhibiting hormone(s), named Gamravali, although its molecular identity is not known (Li, 2004; Matyash, 2004). In Drosophila, ecdysone is for molting and dnpc1a mutants are defective in larval molting owing to ecdysone deficiency. In mammals, neurosteroid is required for proper CNS function and Npc1 mutant mice (Griffin, 2004) have a low level of neurosteroid accompanied with neurodegeneration and neurological dysfunctions, which can be partially suppressed by neurosteroid treatment (Huang, 2005).

Given the evident steroid hormone synthesis defect in dnpc1a flies and the neurosteroid deficiency in Npc1/Npc1 mice, the role of NPC1 in steroid hormone biosynthesis becomes a central mystery. The steroid biosynthesis pathways are generally very similar in vertebrates and insects. In mammals, the substrate, cholesterol, is delivered to the outer mitochondrial membrane and then to the inner membrane. In the insect steroid synthesis pathway, dietary cholesterol is first converted to 7-dehydrocholesterol in the ER, then translocated to the mitochondria by an unknown mechanism (Gilbert, 2002). Once in the inner mitochondrial compartment, the sterol (cholesterol in vertebrates, 7-dehydrocholesterol in insects) is converted into different steroid hormones through a series of enzymatic reactions carried out by P450 enzymes. NPC1 protein has not been observed in mitochondria (Higgins, 1999: Neufeld, 1999; Ko, 2001), so it may function in a delivery process. Together, the current studies and others point in one direction: the need for NPC1 to ensure that sufficient intracellular cholesterol substrate is available for steroid hormone biosynthesis. In NPC mutants, cholesterol accumulates in aberrant endosome or lysosome-like compartments. It is hypothesized that this trapping process may cause or reflect a deficiency of cholesterol in other compartments, such as ER and/or mitochondria, resulting in deficient steroid hormone synthesis (Huang, 2005).

A recent study has shown that mitochondria from whole brain preparations of Npc1 mutant mice contain more cholesterol than similar extracts from wild-type animals (Yu, 2005). This apparent contradiction to the cholesterol deficiency model could be due to pooling different cell types, such as diverse neurons that do not synthesize cholesterol, with cells that do, thus masking a sterol deficiency in the mitochondria of critical cell types. Alternatively, standard mitochondria preparations usually contain some endosomes and lysosomes. The cholesterol richness of endosomes and lysosomes might have masked a cholesterol deficit in mitochondria. The masking may be particularly severe in npc1 mutants as there is massive accumulation of cholesterol in mutant endosomes and lysosomes (Huang, 2005).

Mammals synthesize numerous steroid hormones that have a wide range of important physiological functions. No defect in general steroidogenesis has been observed in NPC1-deficient humans or mice (Soccio, 2004); only neurosteroids are lacking from Npc1 mutant mice (Griffin, 2004). Why is neurosteroid biosynthesis particularly sensitive to the loss of NPC1 function? The answer may come from the source of cholesterol for mitochondria and the route for moving cholesterol to mitochondria. In mammals, the source of cholesterol for mitochondrial steroidogenesis varies between cell type or condition: some cells use HDL cholesterol esters in lipid droplets, others use LDL cholesterol arriving via the endosomal pathway, and still others obtain cholesterol by de novo synthesis from acetate (Soccio, 2004). The differing sources and pathways may make NPC1 more important for steroid synthesis in some cell types than in others (Huang, 2005).

Purkinje neurons, which undergo prominent neurodegeneration in NPC disease, are the main cells for neurosteroid biosynthesis in the brain (Tsutsui, 1999). Using chimeric mice in which some cells have Npc1 function and others do not, it has been shown that Npc1 is required within Purkinje neurons for their survival (Ko, 2005). The apparent cell autonomous role of NPC1 in Purkinje cells raises a question: if NPC1 is required only for neurosteroid biosynthesis, why do wild-type cells fail to help their mutant neighbors? One explanation could be an unidentified function of NPC1 in Purkinje cells in addition to controlling neurosteroid biosynthesis. Alternatively, there may be a cell-autonomous autocrine neurosteroid signal in Purkinje cells (Huang, 2005).

Like mammalian NPC1, Drosophila dnpc1a is widely expressed, but the results of this study show that the crucial function of dnpc1a for development into an adult is restricted to a single tissue (the ring gland), and to a specific biological process (ecdysone biosynthesis) (Huang, 2005).

If the steroid synthesis hypothesis about NPC is correct, the disease should be regarded more as a sterol shortage disease than a sterol excess disease, because the accumulated sterol embedded in multi-lamellar membranes is evidently unavailable for further sterol metabolism. The cholesterol shortage model clearly differs from models in which the lipid accumulation itself is the cause of disease pathology. In fact, individuals with NPC live for many years carrying significant accumulations of lipids, including sterols and gangliosides, in many cells. Adult Drosophila dnpc1a homozygotes, rescued by ring gland dnpc1a expression, seem fully functional except for male sterility, despite the punctate sterol accumulation in many of their tissues. It remains unclear how much damage the accumulated sterol and other lipids impart (Huang, 2005).

The sterol shortage model is also supported by studies of mammalian NPC1 disease, where the transcriptional program for sterol biosynthesis involving the SREBP transcription factor is triggered in cells that are replete with sterol. Normally such sterol abundance would leave SREBP tethered in the ER. SREBP is activated by a regulatory system that senses sterol level in the ER and, when sterol seems low, allows SREBP to move into the Golgi and then to the nucleus where it triggers transcription of genes for sterol synthesis, such as HMG CoA reductase, and genes for sterol import, such as LDL receptor. The activation of SREBP in Npc1 mutant cells, despite the abundant sterol, shows that the accumulated sterol is invisible to the cells' regulatory machinery (Huang, 2005).

These studies suggest possible new directions for improving NPC disease therapy. Previous therapeutic efforts in lowering cholesterol level and limiting dietary cholesterol supply have been unsuccessful (Somers, 2001), as would be expected if the accumulation of sterol in cells is not the primary cause of pathology. Instead, the results point in the opposite direction: the disease might be usefully treated by increasing the cholesterol level in the mitochondria of critical types of neurons (Huang, 2005).

The evolutionary conservation of sterol accumulation in Npc1 mutants of flies and mammals implies a common trafficking function that existed at least half a billion years ago. The further possible similarity in disease mechanisms that is implied by the steroid deficit in Drosophila and mice NPC model may permit advances to be made in understanding mammalian disease by employing classical genetic approaches in flies (Huang, 2005).

Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila

This study shows that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty (Texada, 2022).

Nutrition is one of the most important influences on developmental growth and maturation. Malnutrition or disease can impair growth and delay puberty, whereas obese children enter puberty early. Similarly, Drosophila larvae exposed to poor nutrition, tissue damage, or inflammation delay their development, whereas rich conditions promote rapid growth and maturation. These environmental factors are coupled to the appropriate gating of steroid production via internal checkpoints, one of which is a nutrition-dependent “critical weight” (CW) required to initiate the maturation process (Texada, 2022).

This suggests that signals reflecting nutritional state and body-fat storage play a key role in activating the neuroendocrine pathways that trigger puberty. Although studies suggest that the adipokine leptin may be involved, the mechanisms linking body fat to puberty are poorly defined, and the potential involvement of lifestyles associated with excessive accumulation of cholesterol, one of the most important lipids, has not been considered. In humans, white adipose tissue is the main site of cholesterol storage and can contain over half the body's total cholesterol in obesity (Texada, 2022).

The results show that dietary cholesterol intake promotes systemic body growth through insulin-dependent pathways and that animals raised on high dietary cholesterol initiate maturation earlier. It was show nthat cholesterol is sensed through an Npc1-regulated TOR-mediated mechanism in the fat body and the glial cells of the BBB, which relay information to the IPCs within the brain to promote insulin expression and release, thus coupling growth and maturation with cholesterol status (Texada, 2022).

Insect CW likely evolved as a mechanism ensuring that maturation will not occur unless the animal has accumulated adequate nutrient stores to survive the nonfeeding metamorphosis period and has completed sufficient growth to produce an adult of proper size and thus of maximal fitness. Likewise, the link between body fat and maturation in humans probably ensures adequate stores of fat before maturation onset to support pregnancy and reproductive success (Texada, 2022).

In Drosophila, insulin signaling plays a critical role in coordinating steroidogenesis with nutritional conditions. Insulin acts upon the prothoracic gland (PG) and induces a small ecdysone peak early in L3 that is correlated with CW attainment. In combination with nutrition-related signaling mediated via insulin, nutrient availability is also assessed directly in the PG and is coupled to irreversible endoreduplication that permits ecdysone production at CW (Texada, 2022).

These findings show that accumulation of cholesterol in the PG, induced by the loss of Npc1a, drives a remarkable TOR-dependent increase in endoreduplication and leads to inappropriate attainment of CW. Taken together, these findings indicate that cholesterol sensed by the BBB glia and the fat body promotes growth through insulin signaling and that cholesterol sensed by the PG accelerates maturation through ecdysone signaling. Loss of Npc1 function in humans leads to the Niemann-Pick lysosomal storage disorder, marked by intracellular cholesterol accumulation. Although neurodegeneration is the hallmark of NPC disease, including in a Drosophila model,70 alterations in glial, adipose, hepatic, and endocrine systems are also components of NPC syndrome. In humans, Npc1 itself is strongly expressed in glia and in adipose tissues, especially in obese individuals, and variants in Npc1 are associated with obesity, type-2 diabetes, and hepatic lipid dysfunction (Texada, 2022).

These findings link glial and adipose-tissue cholesterol sensing through Npc1 to systemic growth and metabolic control through effects on insulin signaling. It was also found that intracellular cholesterol accumulation driven by Npc1 loss leads to hyperactivation of TOR that drives increases in DNA replication and cell growth. TOR activity is frequently upregulated in cancer, and the results therefore provide mechanistic insight for understanding the emerging link between cholesterol and a range of cancers. As the coupling of nutrition with growth and maturation is ancient and highly conserved, this work provides a foundation for understanding how cholesterol is coupled to developmental growth and maturation initiation in humans. These findings link a high concentration of this particular lipid in adipose tissues to the neuroendocrine initiation of maturation, which may explain the critical link between obesity (body fat) and early puberty (Texada, 2022).

DEVELOPMENTAL BIOLOGY

As a first step toward unveiling the function of dnpc1a, the temporal and spatial expression of dnpc1a throughout development was determined by in situ hybridization. During embryonic stages dnpc1a is ubiquitously expressed, with higher levels in several tissues. dnpc1a RNA is first found in preblastoderm and blastoderm embryos, reflecting its presence in maternally provided mRNA. After the blastoderm embryo changes from a syncytium to individual cells, the level of dnpc1a RNA increases and becomes further concentrated in the extending germband. The highest expression is seen in the hindgut of fully extended germband embryos. During germband retraction and before dorsal closure, expression in the aminoserosa is detected. Starting at stage 16, strong staining of the putative prothoracic gland cells in the embryo was observed. The prothoracic gland cells of the ring gland are the predominant source of ecdysteroids, the molting hormones, in Drosophila post-embryonic development (Huang, 2005).

In wandering third-instar larvae the dnpc1a RNA level is highest in the prothoracic gland component of the ring gland, and is accompanied by ubiquitous expression in other tissues including brain, garland cells, midgut and imaginal discs. The observed spatial and temporal pattern of expression of dnpc1a suggests that, like NPC1 in mammals, dnpc1a probably functions in all cells. The higher dnpc1a RNA in embryonic and larval ring glands implies that dnpc1a may be involved in the regulation of ecdysteroids that are produced there (Huang, 2005).

Compared to dnpc1a, dnpc1b has a much more restricted expression pattern. dnpc1b mRNA can be detected in midgut and hindgut during late embryonic stages; any other signal is weak to undetectable (Huang, 2005).

EFFECTS OF MUTATION

Many aspects of cholesterol metabolism are well understood, but relatively little is known of the molecular mechanisms by which dietary cholesterol is absorbed by the intestine and trafficked within cells following its acquisition. The related NPC1 and NPC1L1 proteins function in these processes, although the mechanisms by which this protein family promotes cholesterol absorption and intracellular trafficking remain unclear. A genetic approach in Drosophila was used to further explore the functions of the NPC1 gene family. The Drosophila genome encodes two NPC1 homologs designated NPC1a and NPC1b that exhibit 42% and 35% identity to the human NPC1 protein, respectively, and significant but lesser similarity to NPC1L1. The NPC1a gene is ubiquitously expressed and a null allele of NPC1a confers early larval lethality. The recessive lethal phenotype of NPC1a mutants can be partially rescued by a diet of high cholesterol or by including the insect steroid hormone 20-hydroxyecdysone in the diet. These results suggest that NPC1a is required for efficient trafficking of dietary sterols and that reduced ecdysone production is a major consequence of defective sterol trafficking in Drosophila (Fluegel, 2006).

The existence of multiple NPC1 family members in metazoans raises the question of whether NPC1a provides a function that is more similar to one or the other of the vertebrate NPC1 genes, NPC1 and NPC1L1. Several lines of evidence indicate that NPC1a more closely parallels the function of NPC1. (1) The NPC1a gene, like NPC1, appears to be uniformly and abundantly expressed, whereas NPC1L1 appears to be expressed strongly only in the intestine (Altmann, 2004). (2) The finding that NPC1a expression in the ring gland but not midgut is able to rescue the NPC1a mutant phenotype indicates that this factor is not required for absorption of dietary sterols. This conclusion is further bolstered by the finding that NPC1a mutants are not depleted of sterols as would be predicted if NPC1a mutants were unable to absorb dietary sterols; indeed the sterol content of NPC1a mutant larvae is elevated approximately two-fold relative to controls. In contrast, recent work to explore the expression pattern and mutant phenotype of the Drosophila NPC1b gene suggests a close correspondence between the functions of NPC1b and NPC1L1. These experimental findings validate sequence features of the NPC1a and NPC1b proteins that suggest functional correspondence to NPC1 and NPC1L1, respectively (Fluegel, 2006).

While these studies do not directly address the mechanistic nature of the sterol and ecdysone metabolic defects in NPC1a mutants, it is believed that a model whereby NPC1a facilitates intracellular trafficking of sterols is the simplest interpretation of the data given knowledge of the functional roles of NPC1 homologs in other species. In the ring gland this trafficking defect may prevent delivery of sufficient amounts of sterol to sites of ecdysone synthesis, thus resulting in an ecdysone deficiency and the resulting molting and metamorphosis defects observed in NPC1a mutants. The results further suggest that NPC1a is not absolutely required for intracellular sterol trafficking since the requirement for NPC1a can be bypassed by feeding flies excess cholesterol. While the residual intracellular sterol trafficking remaining in NPC1a mutants may be sufficient for most tissues, this does not appear to be true for the ring gland. Experiments are currently underway to directly test these hypotheses (Fluegel, 2006).

Additionally, the possible involvement of sphingolipid trafficking defects is being explored in the NPC1a mutants that would parallel those which have been reported in vertebrate models of NPCD (Zervas, 2001a; Zervas, 2001b; Gondre-Lewis, 2003). Work on the Drosophila NPC1a gene correlates well with previously reported work in C. elegans. The worm genome also possesses two NPC1 homologs, ncr-1 and ncr-2 (Sym, 2000). ncr-1; ncr-2 double mutants have reduced viability, gonadal migration defects, and constitutively form dauer larvae, a dormant life stage specialized for survival under harsh conditions (Sym, 2000; Li, 2004). The phenotypes of ncr-1; ncr-2 double mutants are similar to a class of ligand-binding domain mutants of daf-12, which encodes a nuclear steroid hormone receptor. Li (2004) demonstrated that feeding cholesterol to animals mutant for ncr-1 and ncr-2 is sufficient to suppress the dauer phenotype associated with the loss of both genes. Genetic epistasis analysis also indicates that the ncr genes function upstream of daf-12. These data suggest that the ncr genes deliver sterol precursors for the synthesis of the daf-12 steroid hormone ligand, thus closely paralleling the current results suggesting a perturbation in ecdysone synthesis in Drosophila NPC1a mutants (Fluegel, 2006).

Because NPCD is a neurodegenerative disorder and it was of interest to explore the effects of altered sterol trafficking on neuronal integrity, brain morphology was analyzed in the adult escapers of the hypomorphic NPC1a alleles. However, no substantial alterations were observed in brain morphology in these animals even after aging hypomorphic animals for as long as 40 days. While the lengthy lifespan and lack of detectable phenotypes associated with these adult escaper animals raise the possibility that NPC1a is only required for the efficient production of ecdysone in the larval ring gland, several observations argue against this conclusion. (1) In contrast to the results obtained with adult escaper animals bearing hypomorphic NPC1a alleles, it was found NPC1^a57A homozygous adults rescued by exogeneous dietary cholesterol have a significantly reduced lifespan and that males are sterile. These findings suggest that at least a small amount of NPC1a expression is required during adulthood. (2) Ecdysone titers are extremely low in adults, suggesting that the requirement for NPC1a function at this stage of development involves another undetermined role in the fly. The mechanisms responsible for the shortened lifespan and male sterility of cholesterol-rescued NPC1a null adults are currently being investigated (Fluegel, 2006).

In conclusion, this Drosophila model may be useful for studying the function of NPC1, the mechanisms of intracellular sterol trafficking and the effects of altered sterol trafficking on neuronal integrity. While significant progress has been made in an understanding of NPCD since the cloning of the NPC1 gene, and the membrane topology of NPC1 and its subcellular distribution, as well as at least some of the lipid mistrafficking and metabolic defects associated with loss of NPC1 function are now known, many fundamental questions remain unanswered. In particular, little progress has been made in an understanding of the molecular mechanism by which NPC1 promotes lipid trafficking, and currently nothing is known of the factors that regulate the expression, function, and distribution of NPC1. Moreover, the mechanism by which loss of NPC1 function results in neuronal death remains unclear. The use of a classical genetic approach in Drosophila to address these questions provides a powerful alternative to a purely biochemical or cell biological approach. The NPC1a allelic series described in this work coupled with the finding that simple dietary factors can influence the associated phenotypes should provide a useful starting point for this work (Fluegel, 2006).

To explore the role of dnpc1a in development, double-stranded RNA (dsRNA) interference was used, and loss-of-function mutants were generated. dnpc1a dsRNA-injected embryos developed normally during embryogenesis, but most of them arrested during the first larval stage (Huang, 2005).

The dnpc1a gene is located at cytological band 31B1. A mutation caused by a transposon insertion, KG05670, had been assigned to the pros35 gene, a gene adjacent to dnpc1a and transcribed in the opposite direction. The KG05670 transposon is closer to the 5' UTR of dnpc1a than to pros35. Using KG05670 as a starting strain for imprecise excision, two deletion alleles were generated of dnpc1a that cause N-terminal 182 and 45 amino acid deletions. The alleles are referred to as dnpc1a¹ and dnpc1a². Based on the nature of the deletions, these two mutations are likely to be null alleles of dnpc1a. In situ hybridization with a probe encompassing the coding region detected no signals in those mutant embryos, providing further evidence that they are null alleles (Huang, 2005).

Consistent with the dsRNAi result, flies homozygous for either of the dnpc1a alleles, or trans heterozygous for the combination, died as first-instar larvae. The larval lethality can be fully rescued by ubiquitous expression of a dnpc1a cDNA-yfp fusion construct, confirming that the lethal phenotype is indeed caused by loss of dnpc1a function and not loss of pros35 function. Embryos produced by mothers homozygous for a dnpc1a mutation in their germline cells, and fertilized by dnpc1a mutant sperm, also died during the first-instar larval stage, showing that dnpc1a is essential for larval development but not for embryogenesis (Huang, 2005).

To address whether Drosophila dnpc1a plays a role in cholesterol trafficking like its mammalian homolog NPC1, the distribution of sterol in wild-type and dnpc1a mutant larvae was examined using filipin staining. Filipin stains free 3-ß-hydroxysterols, including ergosterol and cholesterol, so the filipin-staining pattern may reflect the localization of ergosterol, cholesterol and perhaps other sterols. Drosophila is unable to synthesize its own sterol, instead obtaining sterol from its food. Ergosterol is abundant in fungi, yeast and plants, and can substitute for cholesterol to sustain the growth and reproduction of the fly, so ergosterol is likely to be a major sterol source for laboratory flies that live on a yeast-rich medium (Huang, 2005).

Owing to the small size of first-instar larvae, the staining pattern was first examined in large tissues from this stage: Malpighian tubules and midgut. In wild-type animals, the fluorescent filipin signal highlights the lumen of Malpighian tubules and the cell-cell boundaries of the midgut. In dnpc1a mutants, in addition to the normal localization, a punctate pattern of fluorescence was observed inside cells in both tissues, reflecting sterol trapped in aberrant subcellular structures. This subcellular sterol accumulation phenotype is similar to that of mammalian NPC cells, suggesting a conserved role of dnpc1a in intracellular sterol trafficking (Huang, 2005).

At 25°C, wild-type first-instar larvae normally molt to second instar~48 hours after egg laying (AEL), i.e. about 1 day after hatching. Homozygous dnpc1a mutants remained as first-instar larvae for a prolonged period before dying 90-192 hours AEL. One possible cause of the arrested development is a failure to molt. Molting is normally controlled by a pulse of ecdysone, a steroid that serves as the molting hormone. Ecdysone is synthesized from cholesterol that is, in turn, derived from diet sterols (yeast ergosterol/plant sterol). Flies reared on the ergosterol biosynthesis-defective yeast mutant, erg-6, die during a prolonged first instar, similar to that of dnpc1a (Huang, 2005).

Thus, the first-instar arrest of dnpc1a mutants may well be due to a defect in ecdysone production. The high level of dnpc1a transcription normally present in the ecdysone-producing organ, the ring gland, is consistent with this hypothesis. Alternatively, the dnpc1a mutant phenotype may be due to a defect in the response to ecdysone. To distinguish these two possibilities, 20-hydroxyecdysone-feeding experiments were performed. 20-Hydroxyecdysone (20E) converted from ecdysone (E) is the active molting hormone in vivo. If the defect is in ecdysone production, feeding 20E should rescue dnpc1a mutants. By contrast, if the defect is in the response to ecdysone, feeding the larvae 20E would probably not fix the first larvae arrest of dnpc1a mutants. In any case, any rescue accomplished by adding 20E to the food would indicate that a cause of death is inability to molt and possibly a hormone deficiency (Huang, 2005).

Without 20E, 100% of dnpc1a homozygotes die during the first-instar stage. Feeding the dnpc1a mutants 8 µg 20E per gram of medium starting in the early first instar (26 hours AEL) prevents much of the first-instar arrest: 25% of the animals died at first instar, 29% died during the first to second instar transition (with the double pairs of mouth hooks characteristic of that transition), 45% died in the second instar and 2% died in the third instar. If the feeding with 20E was initiated late in the first instar (40 hours AEL), the rescue was similar but weaker. The results indicate that the first instar arrest of dnpc1a mutants is likely to be a consequence of insufficient ecdysone (Huang, 2005).

For insect ecdysone biosynthesis, the substrate cholesterol is first converted to 7-dehydrocholesterol, probably by a microsomal/endoplasmic reticulum (ER)-localized P450 enzyme (Gilbert, 2002). The 7-dehydrocholesterol must translocate to the ring gland mitochondria, and then move into the internal mitochondrial membrane for further chemical modifications that produce ecdysone (Huang, 2005).

The aberrant sterol accumulation and the apparent shortage of cholesterol-derived ecdysone in dnpc1a mutants seem to create a paradox. The cells have abundant, in fact excessive, sterol that should be sufficient for ecdysone biosynthesis. Perhaps the abnormal sterol accumulation leads to a local shortage of sterol precursor available for ecdysone biosynthesis. Alternatively, it could be that sterol accumulation is somehow toxic and inhibits the ecdysone biosynthesis machinery. To distinguish these possibilities, the sterol concentration in the yeast paste was increased. Although the main sterol in yeast paste is ergosterol (~0.3 mg/g), yeast paste medium also contains a trace of cholesterol (~0.6 µg/g). The first-instar arrest of the dnpc1a mutant was significantly suppressed by increasing cholesterol in the food from a trace amount to 0.14 mg/g or 1.4 mg/g (Huang, 2005).

Sterol availability is evidently limiting in dnpc1a mutants, suggesting that the accumulated mass of sterol in the mutant cells is not available for steroid synthesis. A high level of cholesterol added to the media bypasses the sterol defect, perhaps by allowing sterol to reach the endoplasmic reticulum (ER) or mitochondria directly to nourish ecdysone biosynthesis (Huang, 2005).

Ergosterol is able to support the growth and reproduction of Drosophila. Curiously, adding the level of ergosterol to the medium that allowed rescue by cholesterol (1.4 mg/g or 0.14 mg/g) did not have any rescuing activity. This may indicate that cholesterol and ergosterol are moved into or within cells along at least partly different paths, or that the ergosterol is more susceptible to the diversion into aberrant organelles in the mutant cells (Huang, 2005).

7-dehydrocholesterol is the first metabolic product on the path from cholesterol to ecdysone. Feeding dnpc1a mutants with a high level of 7-dehydrocholesterol was even more effective in suppressing the first-instar lethal phenotype of the dnpc1a mutant than cholesterol feeding. A significant percentage of rescued flies even reach adulthood, although they usually died within a day or two after eclosion. By contrast, feeding the dnpc1a mutants with desmosterol, a sterol that can be used to make ecdysone by some insects but not by Drosophila melanogaster (Gilbert, 2002), or with progesterone, a human steroid hormone derived from cholesterol, did not rescue at all (Huang, 2005).

Cholesterol and 7-dehydrocholesterol are much more potent rescuing agents than 20-hydroxyecdysone, suggesting that in the presence of enough proper substrate, dnpc1a mutant larvae were able to synthesis their own ecdysone and quite possibly control the timing and amount of hormone production. 20-Hydroxyecdysone may rescue more poorly because the mutant larvae cannot control the time of exposure, location or amount of ecdysone. The results also indicate that the enzymatic machinery for ecdysone biosynthesis is probably functional in dnpc1a mutants (Huang, 2005).

Since the Drosophila dnpc1a model recapitulates the sterol accumulation phenotypes of NPC disease, whether the flies also have a neurodegeneration problem, another characteristic of mammalian NPC disease, was investigated. Drosophila neurodegeneration mutants often have a short life span and numerous large vacuoles in brain. Although dnpc1a mutants die during the first instar, 7-dehydrocholesterol treatment can extend the mutant lifespan to adulthood. This provided an opportunity to examine the adult brain and search for possible neurodegeneration. Brains from 7-dehydrocholesterol-treated dnpc1a sick adult escapers were sectioned before the escapers died. The gross brain morphology is fine in the mutants and there were no evidence of neurodegenerative vacuoles in the brain sections. To further investigate possible neurodegeneration that might be missed in animals partially rescued with sterol, brains from 96 hour AEL first-instar dnpc1a mutants were examined directly. Again the gross brain morphology was fine in the mutants, with no evidence of typical neurodegenerative vacuoles. The possibility that neurodegeneration may happen in small subset(s) of neurons cannot be excluded (Huang, 2005).

The rescue by ecdysone suggests a hormone deficiency, and the rescue by specific sterols suggests a sterol deficiency. The apparent sterol deficiency could be due to either less sterol uptake from the food, causing a global shortage, or less sterol available for ecdysone production in ring gland cells, causing a local shortage in that tissue. The former possibility seems less likely as abundant sterol accumulates in aberrant organelles in most or all tissues. The 'shortage' of sterol in any tissue, it appears, is mainly a problem of accessing the sterol (Huang, 2005).

Which tissue requires npc1a function in order for normal molting to occur? If dnpc1a is required in tissues where sterol is absorbed from food, such as midgut, the primary defect is probably a global shortage of sterol. If dnpc1a is required within cells that make hormone, then the defect is very likely to be due to a local shortage and the failure to make enough hormone. If dnpc1a is required in tissues that undergo metamorphosis, the primary defect is probably in the response to hormone or in a sterol-related function other than hormone synthesis, or both (Huang, 2005).

The UAS-Gal4 system was used to drive tissue-specific expression of a functional dNPC1a-YFP fusion gene in otherwise dnpc1a mutant flies. Tub-Gal4, a Gal4 driver that activates target genes in all tissues, was combined with UAS-dnpc1a-yfp. This pair of transgenes fully rescued dnpc1a mutants so that they developed into fertile adults, and also prevented abnormal sterol accumulation in all tissues. 69B-Gal4, which drives dNPC1a-YFP expression in ring gland, brain, embryonic epidermis, imaginal discs and testis, also fully restores development of dnpc1a mutants into fertile adults (Huang, 2005).

The most informative experiment came from using Gal4 drivers that produce ring gland-specific expression of dNPC1a-YFP. Either the 2-286 or Feb36 Gal4 driver allowed otherwise dnpc1a mutants to enjoy robust adult viability. The subcellular accumulation of sterol in many tissues other than the ring gland was not reduced under these conditions, providing confirmation that the main ectopic expression of the rescuing gene is in the ring gland. The ring gland is composed of the prothoracic gland (producing ecdysone hormone), the corpora allata (producing juvenile hormone) and the corpora cardiaca. The Feb36 Gal4 driver drives expression in the prothoracic gland and corpora allata. Moreover, a corpora allata-specific Gal4 driver (Aug21) did not provide any rescuing activity, so dnpc1a is required in the ecdysone-producing prothoracic gland cells of the ring gland. By contrast, many non-ring gland GAL4 drivers tested, such as the pan-neural driver, elav-Gal4, the endoderm (midgut)-specific driver 48Y-Gal4 and the muscle-specific driver MHC-Gal4, were not able to rescue the development of dnpc1a mutants beyond first larval instar (Huang, 2005).

These results indicate that dnpc1a is required in the prothoracic gland component of ring gland for ecdysone biosynthesis, supporting the hypothesis that dnpc1a mutants cannot avail themselves of the sterol in the aberrant organelles and therefore cannot make adequate molting hormone. The mutant flies rescued with ring gland expression of dnpc1a provided a good opportunity to examine dnpc1a functions in other tissues at later stages (Huang, 2005).

dnpc1a mutants with ring gland-specific expression of dNPC1a-YFP driven by the 2-286 driver were used to examine phenotypes in third-instar larvae and adults. The tissues examined, brain, imaginal discs, trachea, ovaries, testis and Malpighian tubules, are in some cases too small to dissect and study in detail in first-instar larvae. In all mutant tissues examined, normal filipin staining was seen at cell-cell boundaries and surfaces, plus abnormal sterols accumulated as in mutant first-instar larvae. Sterol accumulation was directly compared in the third instar ring glands and brains from wild type, dnpc1a mutants rescued by ecdysone feeding and dnpc1a mutants rescued by ring gland-specific expression of dnpc1a-YFP. As expected, no sterol accumulated in wild-type ring glands and brains. dnpc1a mutants rescued by ecdysone feeding have sterol accumulation in both ring glands and brains, while dnpc1a mutants rescued by ring gland-specific expression of dNPC1a-YFP have no sterol accumulation in the ring glands but have sterol accumulation in the brains (Huang, 2005).

Among all the mutant tissues examined, Malpighian tubules, which serve a function similar to that of mammalian kidneys, had the most robust sterol accumulation phenotype. To examine the structures of the punctate sterol accumulations at higher resolution, adult Malpighian tubules from wild-type and dnpc1a mutants were analyzed by electron microscopy. Large multi-lamellar structures (0.5-2 µm) were present in mutant Malpighian tubule cells but never in wild-type cells. More than 80% of the multi-lamellar structures were clustered together to form aggregates 1-4 µm across. The multi-lamellar structures are likely to correspond to the sterol accumulation observed in the light microscope after filipin staining. Multi-lamellar structures have been observed in samples from individuals with Niemann-Pick type C mutations. Excess sterol that accumulates because of lipid trafficking defects may be stored in similar aberrant organelles in Drosophila and mammals. The sterol supplementation data suggest that the sterol in those multi-lamellar organelles is not available for synthesizing steroid hormones (Huang, 2005).

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, NPC1, 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 (Flybase term: Niemann-Pick Type C-2, abbreviated NPC2) 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 (CG3153), 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. It is concluded 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 (Huang, 2007).

Cholesterol, an essential component of eukaryotic cell membranes, also serves as the precursor of many steroid hormones and thus plays vital roles in many developmental processes. Cells in the body maintain proper cholesterol levels through elegant homeostatic regulatory systems. Defects in cholesterol homeostasis and metabolism have been linked directly or indirectly to many disease conditions (Huang, 2007).

Niemann-Pick type C (NPC) disease is one such cholesterol homeostasis-related disorder characterized by aberrant accumulation of free cholesterol in late endosome and lysosome-like compartments. Normal cells take up exogenous cholesterol through the receptor-mediated low density lipoprotein (LDL) endocytic pathway. LDL-derived free cholesterol must then leave the endosomal compartment, a process that is blocked in NPC disease cells, to move to other membrane compartments, including the endoplasmic reticulum (ER), and to control homeostatic responses. NPC disease is a progressive neurodegenerative disorder in which the degeneration of cerebellar Purkinje neurons is most prominent. Although the link between cholesterol homeostasis defects and neurodegeneration remains enigmatic, the deficiency of oxysterol and/or neurosteroid has recently been implicated as partially responsible for this neurodegeneration (Huang, 2007 and references therein).

Mutations in either of two different human genes, NPC1 and NPC2, result in Niemann-Pick type C disease, with NPC1 mutations accounting for about 95% of known cases. The large Npc1 protein has 13 transmembrane domains and a sterol-sensing domain (SSD). Npc2, a small, secreted protein that binds cholesterol strongly, was first found as an abundant component of human epididymal fluid and later linked through human genetics to the inherited cause of NPC disease in about 5% of the families studied. The crystal structure of Npc2 has been determined and found to contain a cavity that genetic analyses show to be the likely binding site for cholesterol. Npc2 may serve as a lysosomal cholesterol transporter, rapidly transporting cholesterol to acceptor membranes. Although Npc1 and Npc2 are different types of cholesterol-binding proteins, they appear to be in a common pathway or process based on the virtually indistinguishable phenotypes of the human patients carrying one or the other homozygous mutation (Huang, 2007).

To uncover the disease mechanisms as well as the biological function(s) of NPC proteins, useful NPC disease models have been established in yeast, worms, flies and mice. Drosophila NPC models have been developed using npc1a (also referred to as NPC1- FlyBase) mutations. Drosophila and all other insects are unable to synthesize sterol from simple precursors. In order to synthesize the molting hormone 20-hydroxyecdysone (20E) and to sustain the growth and reproduction of the fly, sterol has to be obtained from food. In Drosophila, npc1a is crucial for sterol homeostasis, as is mammalian NPC1. The fly mutants have a molting defect and homozygotes die as first-instar larvae due to a deficiency of the molting hormone 20E, the primary steroid hormone identified in insects to date. 20E plays crucial roles in insect oogenesis, embryogenesis and metamorphosis. npc1a mutants can be rescued by feeding them excess 20E or either of two of its precursors: cholesterol or 7-dehydrocholesterol. Thus, the ecdysteroid deficiency is evidently due to an inability to access sufficient sterol precursor, a somewhat surprising result given the massive accumulations of sterol in punctated structures that are seen in the mutants by filipin staining. The simplest explanation is that the accumulated sterol, stored in multi-lamellar and multivesicular compartments, is not available for 20E synthesis (Huang, 2007).

Based on the findings in npc1 mutant worms, flies and mice, a cholesterol shortage model of NPC disease has been proposed. The normal function of Npc1 protein may be to promote delivery of sufficient sterol to the ER and/or mitochondria, organelles in which specific steps of steroidogenesis occur. This paper examined the functions of Npc2 proteins in Drosophila. The results further support the cholesterol shortage model (Huang, 2007).

The Npc2 protein has been conserved throughout much of eukaryotic evolution. Only one npc2 gene is present in yeast, worm, mouse and human genomes. Drosophila, clearly a highly advanced organism, has a family of eight npc2-like genes, which have been named npc2a-h. The gene family was identified by BLAST searching with the sequence of human NPC2. Protein sequences of Npc2a-h (CG7291, CG3153, CG3934, CG12813, CG31410, CG6164, CG11314 and CG11315 range from 22% to 36% identical to human NPC2 (Huang, 2007).

Of the eight Npc2-like proteins, Npc2a (also referred to as NPC2 in FlyBase) has the highest sequence identity (36%) and similarity (53%) to human NPC2. Further protein sequence analysis within this protein family reveals that CG3934 (Npc2c), CG12813 (Npc2d) and CG31410 (Npc2e) form a subgroup clustered at cytogenetic locus 85F8 on chromosome III, while CG11314 (Npc2g) and CG11315 (Npc2h) form another subgroup clustered at locus 100A3 on chromosome III. Both groups of genes presumably arose from gene duplication events (Huang, 2007).

Crystal structure determination and mutational analyses have shown that Npc2 has three disulfide bonds and forms a hydrophobic core implicated in cholesterol binding. All six disulfide bond-forming cysteine residues are absolutely conserved in Drosophila Npc2a-h proteins. At other positions shown to be functional in mouse Npc2, Npc2a-h proteins have some variation. For example, F66, V96 and Y100 (amino acid numbers correspond to positions in the mature Npc2 protein without the signaling peptide) of mouse Npc2 are located near the hydrophobic core and are involved in cholesterol binding. V96 is the same or highly similar in seven Drosophila Npc2 proteins except Npc2h, F66 is conserved or replaced by the similar amino acid Tyr (Y) in five Npc2 proteins (not in Npc2f, g and h), and Y100 is conserved or replaced by the similar Phe (F) in six Npc2 proteins (not in Npc2c and f). D72 and K75 of mouse Npc2 are not required for cholesterol binding but are necessary for normal Npc2 function. D72 is conserved or replaced by the related amino acid Glu (E) in four of the Drosophila Npc2 proteins (Npc2a, e, f and h), while K75 is conserved in only three Npc2 proteins (Npc2e, g and h). The variations of these key residues in Npc2 proteins may allow retention of cholesterol-binding ability while adding some capability to bind to sterols other than cholesterol. Evidence for functional conservation despite the changes in key residues is provided by the rescue of mammalian Npc2-mutant cells with an introduced yeast NPC2 gene, which also has changes in encoded key residues such as K75 and Y100 (Huang, 2007).

The npc2-like gene family is present in other sequenced Drosophila genomes, such as D. yakuba, D. pseudoobscura and D. virilis, as well as genomes from other insect species, including Anopheles gambiae (at least eight npc2-like genes), Bombyx mori (at least three npc2-like genes) and Tribolium casteneum (at least three npc2-like genes). Together these data suggest possible multiple rounds of gene duplication events within Class Insecta (Huang, 2007).

The gene structures of Drosophila melanogaster npc2a-h reveal a pattern of evolution in the generation of introns within the coding region. Three genes (npc2a, g and h) have no intron. Two genes, npc2b and d, each have one intron in the same position. Two others, npc2c and e, have two introns in the same positions. The eighth gene, npc2f, has three introns. Interestingly, the intron positions in the Drosophila npc2-like gene family are almost identical to the intron positions of the vertebrate npc2 genes, including those from human, mouse, rat, chimpanzee, cow and zebrafish. By contrast, the intron position in ncr-2, the Caenorhabditis elegans homolog of npc2, is different. Together, the chromosomal clustering of npc2 genes and the similarity of intron positions support the concept that the generation of the npc2 gene family was a result of multiround gene duplication events (Huang, 2007).

To address the potential roles of different NPC2-like proteins, the temporal and spatial expression patterns of the npc2a-h genes during embryonic stages was determined using whole-mount in situ hybridization. The data revealed that npc2a has the broadest expression pattern, whereas other npc2 genes are either not detectably expressed or expressed in restricted locations. The npc2a gene provides a substantial maternal contribution of RNA, and is also ubiquitously expressed at all stages examined. High levels of npc2a expression were found in midgut, salivary gland and ventral nerve cord. npc2b is expressed at the highest levels in the trachea and hypopharynx. npc2g is specifically expressed in head mesoderm and fat body. npc2d and npc2h transcripts could be detected only in salivary gland, while npc2e is expressed in hindgut. The expression of npc2c and npc2f was not detected by in situ hybridization at any time during embryogenesis (Huang, 2007).

Since npc1a is highly expressed in the ring gland, and ring gland expression of npc1a is important for ecdysteroid biosynthesis, the expression of npc2a-h in ring glands was examined. Brains and imaginal discs from wandering third-instar larvae were also examined. In contrast to npc1a, none of the npc2a-h genes was highly expressed in ring glands. Moderate levels of gene expression were detected in larval ring gland , brain and imaginal discs for several npc2 genes, including npc2a and npc2b (Huang, 2007).

Because npc2a has the broadest expression pattern among the eight genes studied, and the highest protein sequence similarity to vertebrate Npc2, focus was initially placed on characterizing npc2a function using mutant phenotypic analysis. Through P element imprecise excision three deletion alleles (npc2a²³⁹, npc2a²⁷¹ and npc2a³⁷⁶ were generated. The whole coding region of npc2a was completely deleted in each of the three alleles, yet homozygous mutant animals were viable and adults were fertile. Each allele was tested in trans to several different genetic deficiencies that remove the gene, and these genetic combinations were also viable and fertile. Whole-mount in situ hybridization with an npc2a antisense probe did not detect any RNA signal in homozygous npc2a mutant embryos, indicating that they are bona fide npc2a mutants (Huang, 2007).

The sterol distribution was next examined in npc2a mutants using filipin staining. Filipin, which stains non-esterified sterols, is often used to study sterol accumulation in NPC1 and NPC2 mutant mammalian cells. Filipin has been successfully used to determine the sterol distribution in Drosophila npc1a mutants and it was found that homozygous mutants have an abnormal sterol distribution similar to that found in mammalian NPC mutants. This is most easily seen by light microscopy as a punctate pattern of filipin-stained particles, and with electron microscopy as multi-lamellar structures (Huang, 2007).

In npc2a/npc2a mutant tissues, including salivary gland, midgut, Malpighian tubules, imaginal discs, brains, trachea and ovaries, a punctate pattern of filipin fluorescence was found. Most tissues had many such spots of accumulated sterol, except trachea, where fewer puncta were found. The filipin staining phenotype was similar to that of Drosophila npc1a mutant tissues and mammalian NPC mutant cells, indicating a conserved role for Drosophila npc2a in regulating efficient intracellular sterol trafficking. The sterol distribution abnormality in npc2a/npc2a mutants could be fully rescued by ubiquitous expression of a UAST-npc2a transgene, indicating that this phenotype is indeed due to npc2a mutation (Huang, 2007).

The structure of mutant npc2a/npc2a cells was examined using electron microscopy. Large multi-lamellar body and multi-vesicular body structures were found in npc2a mutant Malpighian tubules, just as in homozygous npc1a mutants. The multi-lamellar structures were often clustered together to form large inclusions with or without electron-dense materials within. The similarities in cellular phenotypes and ultrastructural defects of npc1a and npc2a mutants further suggest the conserved roles of NPC genes in regulating intracellular sterol trafficking from Drosophila to mammals. As the homozygous mutants survive to adulthood, while npc1a/npc1a flies do not, there must be important differences between npc1 and npc2a phenotypes, and accumulation of sterol is not, by itself, adequate to cause death (Huang, 2007).

The apparently similar defects in sterol distribution in Drosophila npc1a and npc2a mutants raise the question: why do npc1a mutants die as first-instar larvae, while npc2a mutants are viable and ultimately fertile? It was suggested that the first-instar larval lethality of npc1a is due to ecdysteroid deficiency, although this was inferred rather than measured directly. The difference in phenotypes between npc1a and npc2a homozygotes could reflect different ecdysteroid levels (Huang, 2007).

Ecdysteroid levels were directly measured during the first-instar stages (38 hours after egg laying) of wild-type, npc1a/npc1a and npc2a/npc2a larvae. Compared to wild type, the npc1a mutant had low ecdysteroid titers (16.7±0.9 pg/100 mutant larvae versus 87.7±4.4 pg/100 wild-type larvae). The npc2a/npc2a mutant larvae had somewhat lower than normal ecdysteroid levels (53.3±3.6 pg/100 mutant larvae versus 73.8±4.1 pg/100 wild-type larvae). These results could explain why npc1a mutants die as first-instar larvae, i.e., cannot molt, while npc2a mutants are viable and are fertile as adults. Furthermore, the data support the previous hypothesis that the first-instar lethality of npc1a/npc1a mutants is due to ecdysteroid deficiency (Huang, 2007).

Drosophila npc2a and npc2b play redundant roles in regulating sterol homeostasis and 20E biosynthesis. The mutant phenotypes of npc2a; npc2b double-homozygous mutants support the proposed cholesterol-shortage model. Moreover, the apoptotic neurodegeneration observed in the fly mutants suggests a further similarity to mammalian NPC disease, and opens up the possibility of applying model organism genetics to understanding the disease process more completely and perhaps devising treatments (Huang, 2007).

A single gene encoding the cholesterol-binding protein Npc2 is present in many eukaryotic species, with the notable exception that a family of Npc2-like proteins arose within insects or their ancestors. The gene structure analysis of the Drosophila npc2-like gene family clearly indicates that the npc2-like genes were formed by multiple rounds of gene duplication. Why do insects have so many Npc2-like proteins and what are their roles (Huang, 2007)?

In general, gene duplication allows the evolution of new gene functions. In that case, one copy can retain the original function of its ancestor and the other can gain new biological functions through further mutation. The prominent sterol accumulation phenotype in many tissues of the npc2a mutant, the broad expression of npc2a, and the high degree of sequence identity between Npc2a and human NPC2 compared with the other seven Npc2-like proteins, all suggest that npc2a functions similarly to vertebrate npc2. From that perspective, the mystery is about the roles of Npc2b-h. Thise study of npc2b demonstrates that npc2b is especially highly transcribed in trachea, and in that tissue it is partially redundant to npc2a with respect to sterol homeostasis. This is an incomplete answer to the origin of the gene duplications, because it is not clear why two genes are required. Other npc2 genes (npc2c-h) may also function partially redundantly with npc2a because npc2a; npc2b double mutants have a weaker phenotype than npc1a mutants (larval/pupal lethal versus first-instar lethal). Since insects are cholesterol auxotrophs and need external sterol sources for growth, it is possible that some of the Npc2-like proteins may be involved in sterol uptake. The pattern of introns in the Drosophila npc2 gene family provides additional insight into their evolution by suggesting a possible sequence of gene duplication events. The intron-less npc2 genes (npc2a, g, h) may have come first, since the vertebrate genes also lack introns. Next to arise would be npc2 genes like npc2b and d that have a single intron in position 1. An additional intron appears at position 2 in npc2c and e, and the most elaborate gene, npc2f, has a third intron in position 3. Alternatively, the ancient gene may have had three introns, and the other genes have been generated by successive loss of introns. Since the intron positions in vertebrate NPC2 genes are almost identical to those in Drosophila npc2 genes, one can speculate that they were generated in the same order through evolution (Huang, 2007).

As a classical lysosomal storage disease, NPC disease is characterized by the accumulation of large amounts of free cholesterol and other lipids in lysosome-like compartments. The search for the causes of this pathology focused mainly on potential cytotoxic effects caused by the accumulation of cholesterol and other lipids. However, cholesterol-lowering drug treatments did not alleviate NPC disease progression and sometimes made it worse, arguing strongly against the original sterol-excess theory of the disease. To elucidate the molecular and cell biology of NPC protein functions, and shed light on the causes of NPC pathology, NPC models have been established in yeast, worms, flies and mice (Huang, 2007).

These studies of Drosophila npc2 genes are consistent with the sterol-shortage model. In this model, sterols are trapped in aberrant organelles in NPC mutant cells, and therefore insufficient amounts of sterol reach the ER or mitochondria. In mammals, the lack of sufficient sterol in the ER triggers a homeostatic activation of transcription of genes that encode machinery for the synthesis and import of sterol, thus setting in motion a sustaining cycle of excess sterol, leading to more excess sterol. In flies and mice, the failure to bring sufficient sterol substrate to the ER/mitochondria could deprive cells of the ability to synthesize adequate steroid hormone. The consequences are different between mammals and flies, because the actions of steroids are quite different. In flies the principal steroid hormone is 20E, the molting hormone, so the defect is a failure to molt. In mammals the cerebellar Purkinje neurons are known to produce multiple neurosteroids, although their functions are far from clear. Npc1/Npc1 mutant mice are deficient in neurosteroids, and administration of supplementary allopregnanolone reduces the symptoms of NPC disease. Thus, both fly and mouse NPC mutants are steroid hormone deficient and both mutants can be rescued by exogenous steroid hormone treatment, suggesting strongly that cholesterol and the consequent steroid shortages play a central role in NPC disease (Huang, 2007).

These studies reveal a new layer of ecdysteroid biosynthesis regulation, i.e. sterol substrate availability. The regulation of ecdysteroid biosynthesis and the downstream events that mediate ecdysteroid hormone action have been studied continuously for several decades using genetic and biochemical approaches. To date, many genes that affect 20E biosynthesis have been identified and characterized, and these can be grouped into four functional classes. The first class of genes includes upstream factors such as prothoracicotropic hormone (PTTH) that control whether the prothoracic gland should synthesize ecdysone or not. A PTTH mutant has not been isolated in Drosophila, but studies in other insects have clearly demonstrated the essential function of PTTH in ecdysteroid biosynthesis. The larval arrest phenotypes resulting from ablating Drosophila neurons that produce PTTH are consistent with a role in governing ecdysteroid biosynthesis. The second class of genes consists of the yet-to-be-identified PTTH receptor and the Ras signaling cascade that transduces the PTTH signal. Ras appears to act through its downstream effector Raf to control ecdysteroid biosynthesis. The third class of genes includes nuclear transcription factors and regulators, such as ftz-f1, ecd, woc and mld. The targets of these proteins are not well defined but may include the fourth class of genes, the Halloween genes (e.g. dib, sad, phm, shd, spo and spo2) that encode p450 enzymes that mediate the conversion of cholesterol to 20E through multi-step reactions in the ER and mitochondria (Huang, 2007).

The present study, together with study of Drosophila npc1a, defines a fifth class of genes functioning to ensure a sufficient supply of sterol substrates for 20E biosynthesis. This class of mutants has intact 20E biosynthetic enzymes, as shown indirectly by feeding and rescue experiments, but has insufficient sterol substrate for 20E production. Therefore, the ecdysteroid-deficient mutant phenotype can be suppressed by excess cholesterol or 7-dehydrocholesterol, as in npc1a or npc2 (a and b) mutants. Other members of this gene class may include some START domain-containing proteins as well as PBR, which are implicated in transporting sterol into mitochondria for steroid biosynthesis in mammals (Huang, 2007).

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

The Niemann-Pick type C1 (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. This study shows 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 (Voght, 2007).

The genomes of humans, mice, flies, and worms each encode a pair of closely related NPC1 homologs. While this finding raises the possibility that the NPC1 paralogs in metazoans have partitioned into specific evolutionarily conserved functional roles, molecular and functional studies have not completely supported this conclusion. For example, worms, flies, mice, and humans each have one NPC1 family member that appears to promote intracellular sterol and lipid trafficking in a broad set of tissues and another NPC1 family member that is expressed in a more restricted fashion. However, the expression pattern of the more restricted NPC1 family member is not conserved. In particular, murine NPC1L1 is expressed predominantly or exclusively in the intestine, where it functions in sterol absorption. By contrast, human NPC1L1 is expressed most highly in liver, suggesting a hepatic function for this protein in addition to its well-documented role in dietary cholesterol absorption. An even greater difference is seen in the expression of C. elegans NCR-2, which is restricted to a pair of neuroendocrine cells and the somatic gonad. The current study indicates that Drosophila NPC1b is exclusively required in the midgut, where it plays a major role in the absorption of dietary sterols. These features most closely resemble those of murine NPC1L1 and, together with previous work, strongly suggest that Drosophila NPC1a and NPC1b provide intracellular sterol trafficking and sterol absorption functions that are equivalent to vertebrate NPC1 and NPC1L1, respectively (Voght, 2007).

The finding that Drosophila NPC1b provides a function that is apparently equivalent to vertebrate NPC1L1 afforded the opportunity to address conflicting models regarding the role of NPC1L1 in cholesterol absorption. Previous work has generated disagreements on the subcellular distribution of NPC1L1: Some studies suggest that it localizes to the plasma membrane, others suggest that it localizes to internal endosomal compartments, and still others suggest that it translocates between compartments as part of a sterol transporter activity. These findings raise questions as to whether NPC1L1 promotes an early step in sterol absorption at the plasma membrane and/or a later step in the intracellular trafficking of sterol-rich endocytic trafficking intermediates. Analysis of midgut tissues from NPC1b mutants supports a model in which NPC1b promotes an early role in the absorption of sterols at the plasma membrane. In particular, the findings that the midgut epithelium from NPC1b mutants appears to be devoid of intracellular accumulations of sterol in transport organelles and that the midgut epithelium is largely depleted of sterol suggest that NPC1b acts at an early step in sterol absorption. However, the possibility cannot be excluded that NPC1b plays an additional role in promoting the intracellular trafficking of sterol-enriched transport organelles following its early role in sterol absorption. Given the similar biological functions of NPC1b and NPC1L1, it is propose that NPC1L1 also acts at an early step in sterol absorption within the small intestine (Voght, 2007).

While the current data suggest that the Drosophila NPC1a and NPC1b genes provide functions equivalent to the vertebrate NPC1 and NPC1L1 genes, respectively, the consequences of mutations in these genes are not equivalent in flies and vertebrates. In particular, mutations in the Drosophila NPC1 family members result in recessive lethal phenotypes at an early stage of development. In the case of NPC1a, this lethality is due to a failure in cholesterol trafficking, leading to decreased production of ecdysone, the steroid hormone responsible for molting and metamorphosis in insects. Null mutations in the vertebrate NPC1 gene also lead to a failure in cholesterol trafficking and severe phenotypes in youth, but they do not lead to defects in endocrine steroidogenesis, as evidenced by the fact that mutations in the murine NPC1 gene do not affect the concentrations of several major circulating steroid hormones. These differences likely reflect the fact that insects are sterol auxotrophs, whereas vertebrates are able to synthesize sterols from acetate and therefore rely less on dietary cholesterol, and perhaps the efficiency of intracellular sterol trafficking. The absolute requirement for sterols in the Drosophila diet may also offer an explanation for the recessive lethal phenotype of Drosophila NPC1b mutants relative to the lack of effect of NPC1L1 mutations on adult viability in vertebrates. However, as discussed more fully below, the effects of NPC1b mutations on viability are not fully understood at present and will require further work (Voght, 2007).

This work on Drosophila NPC1a and NPC1b indicates that these proteins play nonredundant and noninterchangeable roles. In particular, the intracellular sterol trafficking defects seen in NPC1a mutants are not observed in NPC1b mutants, and the sterol absorption defect of NPC1b mutants is not a feature of the NPC1a mutants. Moreover, NPC1b;NPC1a double mutants do not appear to have more severe intracellular sterol trafficking and sterol absorption defects than the NPC1a and NPC1b single mutants, respectively. Transgenic experiments further demonstrate that NPC1a cannot substitute for NPC1b (data not shown) and that NPC1b cannot substitute for NPC1a. The noninterchangeable feature of the NPC1a and NPC1b proteins may derive from differences in the subcellular localization of these factors, the requirement for other tissue- and NPC1-specific cofactors, or the possibility that these factors influence sterol trafficking in unique ways. Further experiments will be required to distinguish these possibilities (Voght, 2007).

Although the sterol absorption assay was primarily developed to explore the function of NPC1b, valuable insights into the regulation and mechanism of sterol absorption were also revealed in sterol absorption studies of NPC1a mutants. For example, the results indicate that loss of NPC1a activity results in significantly increased sterol absorption. This finding is consistent with previous work demonstrating that the sterol content of NPC1a mutants exceeds that of wild-type controls (Fluegel, 2006) and indicates that the increased sterol content of NPC1a mutants derives from increased sterol absorption as opposed to an alteration in sterol turnover. Surprisingly, the upregulation of sterol absorption that occurs in NPC1a mutants appears to be epistatic to the sterol absorption defect of NPC1b mutants, as evidenced by the finding that NPC1b;NPC1a double mutants absorb cholesterol far more efficiently than NPC1b single mutants. These findings indicate that, while NPC1b normally plays an important role in sterol absorption, there is an NPC1a- and NPC1b-independent mechanism of sterol absorption in Drosophila that functions efficiently in the absence of these factors. Interestingly, the lack of sterol enrichment of midgut tissues that is seen in NPC1b mutants is also observed in NPC1b;NPC1a double mutants, suggesting that the NPC1a- and NPC1b-independent sterol absorption pathway somehow bypasses sterol enrichment of midgut tissues. One possible explanation of this finding is that the mechanism of the NPC1a- and NPC1b-independent sterol absorption pathway is fundamentally different from that of the NPC1b-dependent pathway and does not require or facilitate sterol enrichment of midgut tissues. Alternatively, the NPC1a- and NPC1b-independent sterol absorption pathway may operate in tissues other than the midgut, such as Malpighian tubules. Identification of the NPC1a- and NPC1b-independent sterol absorption apparatus will be required to resolve these matters (Voght, 2007).

There are several potential models to explain the effects of mutations in NPC1a on sterol absorption. One possibility is that there is a sterol-sensing mechanism in Drosophila that monitors the flux of sterol trafficking intermediates or the abundance of downstream products of sterol trafficking, such as ecdysone. The sensing mechanism then relays this information to regulate sterol absorption through the alternate sterol absorption pathway. Alternatively, loss of NPC1a activity may decrease the efflux of dietary-derived sterols by blocking the trafficking of sterols to the midgut epithelium plasma membrane for delivery to ATP-binding cassette (ABC) family of efflux transporters. These different modes of regulation may act through a cell-autonomous or non-cell-autonomous mechanism. These models should be readily testable in future experiments (Voght, 2007).

While the findings indicate that NPC1b promotes an early step in dietary sterol acquisition, several observations suggest that NPC1b mutant larvae die for reasons unrelated to sterol deficiency. (1) Filipin staining of the CNS and Malpighian tubules of NPC1b mutants revealed fluorescence intensities comparable to that of wild-type controls, and the results of a conventional assay to measure total sterol abundance indicated that NPC1b mutants are not severely depleted of sterols. (2) Unlike the NPC1a recessive lethal phenotype, which is strongly influenced by dietary sterols, the NPC1b mutant lethal phase is not detectably influenced by increased dietary sterol content. Similarly, increased maternal loading of sterols during oogenesis does not shift the lethal phase of NPC1b mutants, in contrast to the results with NPC1a mutants. (3) In contrast to findings in NPC1a mutants, exogenous ecdysone is unable to alter the lethal phase of NPC1b mutants. This latter finding cannot be readily explained by a defect of NPC1b mutants in the absorption of this nutrient from the midgut because previous studies have shown that 20-hydroxyecdysone can be absorbed directly, apparently without need of active transport. Based on these findings, it is suggested that, while NPC1b promotes the absorption of sterols from the midgut epithelium, the NPC1b gene is essential for viability either because defective sterol absorption in the midgut adversely influences an essential process that is unrelated to ecdysone metabolism or because NPC1b mutants fail to absorb another unknown essential dietary factor. Further studies will be required to resolve this matter (Voght, 2007).

In conclusion, the results indicate that the NPC1b protein normally plays an early role in the acquisition of dietary sterols in the midgut epithelium. However, under conditions of defective intracellular sterol trafficking and possibly dietary restriction of sterols, the absorption of sterols increases, apparently through an NPC1a- and NPC1b-independent mechanism. Moreover, these studies raise the possibility that NPC1b also promotes the absorption of one or more essential nonsterol nutrients. While the work advances current understanding of the molecular mechanisms of sterol absorption, it also raises many new questions -- in particular, the possibility of a mechanism by which the efficiency of intracellular trafficking is monitored in insects and conveyed to a novel NPC1-independent sterol absorption apparatus. The knowledge of the precise mechanism by which NPC1a and NPC1b promote intracellular sterol trafficking and sterol absorption, respectively, and the factors that regulate the expression and function of NPC1a and NPC1b is also far from complete. Further analyses of Drosophila NPC1a and NPC1b mutants should provide answers to these questions (Voght, 2007).

Neuronal loss of Drosophila NPC1a causes cholesterol aggregation and age-progressive neurodegeneration

The mistrafficking and consequent cytoplasmic accumulation of cholesterol and sphingolipids is linked to multiple neurodegenerative diseases. One class of disease, the sphingolipid storage diseases, includes Niemann-Pick disease type C (NPC), caused predominantly (95%) by mutation of the NPC1 gene. A disease model has been established through mutation of Drosophila NPC1a (dnpc1a). Null mutants display early lethality attributable to loss of cholesterol-dependent ecdysone steroid hormone production. Null mutants rescued to adults by restoring ecdysone production mimic human NPC patients with progressive motor defects and reduced life spans. Analysis of dnpc1a null brains shows elevated overall cholesterol levels and progressive accumulation of filipin-positive cholesterol aggregates within brain and retina, as well as isolated cultured brain neurons. Ultrastructural imaging of dnpc1a mutant brains reveals age-progressive accumulation of striking multilamellar and multivesicular organelles, preceding the onset of neurodegeneration. Consistently, electroretinogram recordings show age-progressive loss of phototransduction and photoreceptor synaptic transmission. Early lethality, movement impairments, neuronal cholesterol deposits, accumulation of multilamellar bodies, and age-dependent neurodegeneration are all rescued by targeted neuronal expression of a wild-type dnpc1a transgene. Interestingly, targeted expression of dnpc1a in glia also provides limited rescue of adult lethality. Generation of dnpc1a null mutant neuron clones in the brain reveals cell-autonomous requirements for dNPC1a in cholesterol and membrane trafficking. These data demonstrate a requirement for dNPC1a in the maintenance of neuronal function and viability and show that loss of dNPC1a in neurons mimics the human neurodegenerative condition (Phillips, 2008).

Ninety-five percent of NPC disease cases are caused by mutation of NPC1, a 13-pass transmembrane protein that resides in a unique class of endosomal organelles, binds cholesterol, and has the hallmarks of a transporter involved in sphingolipid/cholesterol trafficking. Loss of the Drosophila ortholog dNPC1a has been reported to result in early lethality attributable to the loss of cholesterol-dependent ecdysone production, a steroid hormone required for molting. This insect-specific requirement has been a distraction, and has prevented detailed characterization of the Drosophila model. Fortunately, this early block in development is easily bypassed through a diet of excess ecdysone precursors (cholesterol or 7-dehydrocholesterol) or by targeted expression of dNPC1a in the ring gland, the endocrine organ that produces ecdysone. Null dnpc1a mutants rescued to adulthood show progressive locomotor defects, greatly reduced life span, intracellular accumulation of cholesterol aggregates, and age-progressive neurodegeneration, a constellation of phenotypes that closely mimic the human NPC disease condition. Importantly, all of these phenotypes are rescued by targeted neuronal expression of dNPC1a in the null mutant, demonstrating a neural requirement (Phillips, 2008).

Previous reports conclude that loss of dNPC1a does not cause neurodegeneration. In contrast, the current study shows that the lack of dNPC1a within the brain causes the age-progressive accumulation of massive intracellular membranous structures, which are never observed in wild-type neurons, and brain degeneration starting as small vacuoles within the retina and progressing to massive tissue loss within both the retina and central brain. Similar neurodegeneration occurs in dnpc1a null mutants fed high levels of cholesterol during larval growth or expressing wild-type dnpc1a in the ring gland. Thus, neither cholesterol feeding nor ecdysone function contributes to the dnpc1a neurodegeneration phenotypes. In Drosophila, dNPC1a-dependent steroid hormone expression clearly explains the requirement for dNPC1a during development. Similarly in mammals, loss of NPC1 is associated with lower neurosteroid levels, and administration of the neurosteroid allopregnanolone reportedly delays onset of neurological symptoms in NPC1^-/- mice. These data clearly argue for NPC1-dependent generation of cholesterol-derived signaling steroids. However, characterized steroid hormones in Drosophila derive from the ring gland, and this study shows that dNPC1a function in the ring gland provides no protection against neurodegeneration (Phillips, 2008).

Targeted neuronal expression of wild-type NPC1 in both the murine and Drosophila models prevents neurodegeneration associated with NPC1 dysfunction. The elav-GAL4-driven expression of wild-type dNPC1a does not lead to glial cell transgene expression. The neurodegenerative process in NPC cases has been linked to glial cell and astrocyte cellular dysfunction because of NPC1 localization to these cell types. However, in the neuronal dNPC1a rescue animals, glial cells lacking dNPC1a adjacent to neurons expressing wild-type dNPC1a still accumulate massive MLBs and filipin-positive puncta. It is presently not known whether these glial cells die. Nevertheless, targeted glial expression of dNPC1a clearly provides significant rescue for the early-onset lethality of dnpc1a mutants, demonstrating that dNPC1a function in glia plays a substantial role in this disease model. Thus, in Drosophila dNPC1a function in both glia and neurons is important for prolonged survival during aging (Phillips, 2008).

Chimeric mice with functional dNPC1 expressed in a few cells still manifest death of nearby npc1 mutant neurons, at least suggesting a cell-autonomous role for NPC1 in neuronal cell survival. However, in Drosophila, MARCM clonal results show that MB neurons apparently do not require dNPC1a for long-term survival. MARCM analyses of aged day 50 dnpc1a null neurons show intact neuronal perikarya, elaborate dendrites, and structurally mature axons. These data, along with the neurodegenerative delay associated with neurosteroid application to npc1^-/- mutants early in murine development, argue for a non-cell-autonomous role for NPC1 function. In contrast, MARCM analysis of randomly induced dnpc1a null clones throughout the brain clearly demonstrates a cell-autonomous accumulation of cholesterol aggregates, as revealed by filipin staining, and the formation of extensive MLBs in neuronal soma, characteristic of ailing cells. It remains formally possible that steroids produced by neurons depend on dNPC1a function and that such neurosteroids maintain neuronal viability and so guard against complete neurodegeneration. Furthermore, although almost all neurons in the NPC^-/- mice are filipin positive, not all neurons are equally vulnerable, because there are low levels of neuron loss in the thalamous and prefrontal cortex in the face of nearly complete PC loss. Similarly, in the Drosophila brain, it appears that some neuron populations are also more sensitive to the loss of dNPC1a, at least at the level of full cellular death (Phillips, 2008).

Loss of dNPC1a function in Drosophila neurons strongly impacts cholesterol trafficking. Null dnpc1a brains accumulate highly elevated levels of cholesterol and cholesterol aggregates at exceedingly high levels within aberrant neuronal organelles. These MLB structures are often composed of hundreds of layers of wrapped membrane and accumulate progressively in both size and abundance in all neurons in the absence of dNPC1a. Whereas the existence and formation of MLBs is not always a pathological symptom, the massive accumulation of MLBs seen in a variety of disease conditions is clearly an indication of severe cellular dysfunction. It has been proposed that sphingolipid storage disease cells upregulate a Beclin-1-regulated autophagic process (self-digestion) to promote cell survival. The formation of cholesterol-rich MLBs seen in NPC1-deficient cells is consistent with such autophagic cell death (Phillips, 2008).

The cytoplasmic accumulation of MLBs in neurons is observed in a variety of other Drosophila mutants, also associated with adult lethality, neurodegeneration, and synaptic dysfunction. The formation of MLBs in the retina and neuromuscular synaptic junction are neuronal phenotypes in the benchwarmer (bnch) mutant, caused by loss of a predicted lysosomal sugar carrier (Dermaut, 2005). The eggroll mutant exhibits similar MLB structures in neurons and glial cells, with associated brain vacuolization and early-onset lethality (Min, 1997). A Drosophila disease model for infantile neuronal ceroid lipofuscinoses (Ppt1 mutants) displays the same accumulation of MLBs throughout the brain, with associated early lethality but without reported signs of brain vacuolization or neuronal tissue loss (Hickey, 2006). The genetic lesion causing eggroll has yet to be determined, but dnpc1a, bnch, and Ppt1 are all lysosomal/endosomal proteins, clearly linking neuronal MLB formation with endosomal trafficking defects (Phillips, 2008).

Sterols (ergosterol and cholesterol) together with sphingolipids are the major components of lipid rafts. Drosophila lipid rafts are important for the light-induced recruitment of INAD-signaling phototransduction complexes in photoreceptors (Sanxaridis, 2007) and also act as positive regulators of the Drosophila metabotropic glutamate receptor (DmGluRA) signaling between neurons. Drosophila lipid rafts also regulate voltage-gated ion channel signaling and the synaptic vesicle cycling underlying neurotransmission. Altering cholesterol levels and trafficking within neurons presumably has a great impact on membrane lipid rafts and their dependent mechanisms. Loss of NPC1 is linked to the depletion of lipid rafts in endocytic membranes. Loss of dNPC1a causes profound changes in cholesterol distribution and thus likely also disrupts lipid rafts. This study shows that loss of dNPC1a disrupts relevant neuronal processes, including phototransduction in retinal photoreceptors and synaptic transmission to the optic lamina. The Drosophila NPC1 model is a good system to investigate whether NPC1 regulates formation or maintenance of lipid rafts in neurons, which may in turn be required for neuronal function and viability. Genetic and proteomic screens can tease out other players in the lipid trafficking pathway and thus provide insight into the dysfunction driving intracellular cholesterol/sphingolipid accumulation and identify potential drug targets for alleviating the catastrophic consequences of the NPC disease (Phillips, 2008).

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann-Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. This study shows that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, it was found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol (7-dC) can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants are enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, this study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis (Wang, 2011).

This study shows that the sterol trafficking protein NPC1 is important for the individualization process in Drosophila spermatogenesis. Although there is sterol accumulation in npc1 testes and the male sterility of npc1 mutants can be rescued by 7-dC supplements, individualization defects are not due to ecdysone shortage, the primary cause of the npc1 mutant larval lethal phenotype (Wang, 2011).

This report provides insight into how NPC1 is involved in the spermatogenesis process. NPC1 and NPC2 are reported to be involved in sterol trafficking in different model organisms. The first hint that NPC genes are important for male reproduction was the discovery of NPC2, which is highly enriched in epididymal fluid. The Drosophila npc1 model provides the first in vivo link between NPC1 and male infertility. Defective sperm development was later described in a mouse npc1 model, but the underlying mechanism is still not clear. This study in Drosophila adds more details about the role of NPC1 in spermatogenesis. First, the function of NPC1 in spermatogenesis is specifically required in the germ cells but not in the somatic cells, suggesting that NPC1 has a cell-autonomous function in this process. Second, NPC1 function is required in the individualization process. Disrupted ICs, leaky caspase activity and scattered nuclei indicate that cooperative aspects of the individualization process are defective. Third, it was shown that individualization defects are likely caused by sterol deficiency but are not due to ecdysone shortage. Therefore, npc1 mutant sterility is due to other sterol-involved defects. It is not surprising that npc1 mutants show phenotypes independent of steroid hormone. Previous studies have shown that npc1 mutants were reported to have progressive neurodegeneration and this defect cannot be rescued by the ecdysone precursor 7-dC, implying a 7-dC, and probably ecdysone, independent mechanism (Wang, 2011).

Many genes have been identified to be involved in the individualization process. However, the functions of most of them are still unclear. So far, actin assembly, apoptosis and membrane trafficking are the three best characterized processes that are known to be important for different aspects of individualization. The individualization complex (IC) is formed by a cohort of 64 actin cones. Therefore, the formation of actin cones, actin dynamics and the integrity of the IC are all essential for individualization. Removal and clearance of excess cytosol is the ultimate goal of individualization. Apoptosis has been shown to be essential for the removal of excess cytosol and this requirement is likely to be conserved in mammals. Lastly, individualization is a rapid membrane-remodeling process. Cog5 and syx5 function in the individualization process by affecting membrane trafficking (Farkas, 2003; Xu, 2002; Wang, 2011 and references therein).

Which process during individualization are sterols required for? Though NPC1 proteins are highly conserved in sequence and npc mutants in different animal models have sterol trafficking defects, global mutant phenotypes differ greatly. An evolutionally conserved mechanism of NPC pathology has emerged from studies on NPC1 mutants in the worm, fly and mouse. Shortage of available sterols, which in turn leads to shortage of cholesterol-derived steroid hormones, appears to be the source of defects in NPC models. Are the individualization defects observed in Drosophila npc1 mutants caused by the same mechanism? Consistent with the sterol trafficking defect observed in the npc1 testis, adding 7-dC rescues male sterility in npc1 mutants, indicating that the sterility is likely due to sterol shortage. However, it was found that although EcRA is expressed in spermatocytes, neither EcR (for ecdysone signaling) nor disembodied (dib), encodes an important P450 enzyme involved in ecdysone biosynthesis, is required in the individualization process. These results indicate that the steroid hormone ecdysone is not required (Wang, 2011).

There are two possibilities for the requirement of sterols in individualization. First, instead of ecdysone, another unidentified steroid hormone is required for individualization. If this is true, since npc1 is required in the germ cell, this steroid must act in a paracrine or autocrine manner to affect individualization. Alternatively, sterols may be intrinsically important for individualization. The individualization process requires membrane bending and remodeling, as well as a large amount of membrane addition. Sterols are key membrane components and sterol content can affect membrane bending rigidity/curvature and fluidity. It is possible that abnormal sterol distribution and sterol deficiency in npc1 mutants may lead to impaired membrane trafficking or membrane-remodeling. Recently, it was found that sterol-rich structures are present in the leading edge of the IC and that, in mutants of the sterol binding protein OSBP, sterol-rich structures are absent and the individualization process is defective (Ma, 2010). Adding cholesterol or 7-dC can rescue male infertility in osbp mutants, indicating that sterility is caused by the sterol deficiency (Ma, 2010). Interestingly, the OSBP-rich or sterol-rich puncta are present in npc1 mutants, suggesting that npc1 may act independent or downstream of Osbp in individualization. Moreover, a temperature-sensitive feature of the individualization defects of npc1 mutants was observed, implying a membrane defects in npc1 mutants. Taken together, this work offers a new avenue for studying how sterols play a role in the individualization process (Wang, 2011).

In summary, this study has reported the male sterile phenotype of the npc1 mutant fly in detail and has successfully connected the NPC1 protein with the spermatogenesis process. These studies in the Drosophila npc1 model may supply valuable clues for future research not only in Drosophila but also in other model systems. During these studies on rescuing both lethality and male infertility, it was noticed that 7-dC usually yields better rescuing results than cholesterol. Exploiting the difference between 7-dC and cholesterol in rescue efficiency may be a useful therapeutic strategy for the treatment of NPC disease. New drugs or treatments that improve the efficiency of delivery and usage of sterols and steroid hormones may have better therapeutic effects (Wang, 2011).

EVOLUTIONARY HOMOLOGS

A yeast Niemann-Pick model

The Niemann Pick C1 protein localizes to late endosomes and plays a key role in the intracellular transport of cholesterol in mammalian cells. Cholesterol and other lipids accumulate in a lysosomal or late endosomal compartment in cells lacking normal NPC1 function. Other than accumulation of lipids, defects in lysosomal retroendocytosis, sorting of a multifunctional receptor and endosomal movement have also been detected in NPC1 mutant cells. Ncr1p is an ortholog of NPC1 in the budding yeast Saccharomyces cerevisiae. This study shows that Ncr1p is a vacuolar membrane protein that transits through the biosynthetic vacuolar protein sorting pathway, and that it can be solubilized by Triton X-100 at 4°C. Using well-established assays, it was demonstrated that the absence of Ncr1p has no effect on fluid phase and receptor- mediated endocytosis, biosynthetic delivery to the vacuole, retrograde transport from endosome to Golgi and ubiquitin- and nonubiquitin-dependent multivesicular body sorting. It is concluded that Ncr1p does not have an essential role in known endocytic transport pathways in yeast (Zhang, 2004).

Niemann-Pick disease type C (NP-C) is a progressive, ultimately fatal, autosomal recessive neurodegenerative disorder. The major biochemical hallmark of the disease is the endocytic accumulation of low-density lipoprotein-derived cholesterol. The majority of NP-C patients have mutations in the Niemann-Pick type C1 gene, NPC1. This study focuses on the Saccharomyces cerevisiae homolog of the human NPC1 protein encoded by the NCR1 gene. Ncr1p localizes to the vacuole, the yeast equivalent to the mammalian endosome-lysosome system. This study identifies the first phenotype caused by deletion of NCR1 from the yeast genome, resistance to the ether lipid drug, edelfosine. The results indicate that edelfosine has a cytotoxic, rather than cytostatic, effect on wildtype yeast cells. The edelfosine resistance phenotype was exploited to assess the function of yeast Ncr1 proteins carrying amino acid changes corresponding to human NPC1 patient mutations. One of these amino acid changes severely compromises Ncr1p function as assessed using the edelfosine resistance assay. These findings establish S. cerevisiae as a model system that can be exploited to analyze the molecular consequences of patient mutations in NPC1 and provide the basis for future genetic studies using yeast (Berger, 2005).

A C. elegans Niemann-Pick model

Niemann-Pick type C (NP-C) disease is a progressive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in lysosomes. 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, which encodes a protein implicated in the retrograde transport of sterols and other cargo from lysosomes. Furthermore, localization of the NPC1 protein to lysosomal/endosomal compartments is essential for proper transport. To create a model of NP-C disease in a simple, genetically tractable organism, deletion mutations were generated in two C. elegans homologs of the human NPC1 gene, designated npc-1 and npc-2. Animals mutant for npc-1 develop slowly, lay eggs prematurely, and are 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 (Sym, 2000).

Mutations in the human NPC1 gene cause most cases of Niemann-Pick type C (NP-C) disease, a fatal autosomal recessive neurodegenerative disorder. NPC1 is implicated in intracellular trafficking of cholesterol and glycolipids, but its exact function remains unclear. The C. elegans genome contains two homologs of NPC1, ncr-1 and ncr-2, and an ncr-2; ncr-1 double deletion mutant forms dauer larvae constitutively (Daf-c). The phenotypes of ncr single and double mutants were analyzed in detail, and the ncr gene expression patterns were determined. The ncr genes were found to function in a hormonal branch of the dauer formation pathway upstream of daf-9 and daf-12, which encode a cytochrome P450 enzyme and a nuclear hormone receptor, respectively. ncr-1 is expressed broadly in tissues with high levels of cholesterol, whereas expression of ncr-2 is restricted to a few cells. Both Ncr genes are expressed in the XXX cells, which are implicated in regulating dauer formation via the daf-9 pathway. Only the ncr-1 mutant is hypersensitive to cholesterol deprivation and to progesterone, an inhibitor of intracellular cholesterol trafficking. These results support the hypothesis that ncr-1 and ncr-2 are involved in intracellular cholesterol processing in C. elegans, and that a sterol-signaling defect is responsible for the Daf-c phenotype of the ncr-2; ncr-1 mutant (Li, 2004).

Identification and structural analysis of Niemann-Pick C1 protein

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA result in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (Carstea, 1997).

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. The data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, the data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important (Davies, 2000a).

Niemann-Pick C1 protein interaction with cholesterol

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. Photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 was performed by using [³]7,7-azocholestanol ([³H]AC). After immunoprecipitation, ³H-labeled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, it was shown that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [³H]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. A procedure was developed to label the NPC1-YFP by [³H]AC in vitro and it was shown that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1 (Ohgami, 2004).

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. It is proposed that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation (Malathi, 2004).

Targeting of NPC1 to late endosomes

Niemann-Pick type C (NPC) disease is a severe cell lipidosis characterized by the accumulation of unesterified cholesterol in the endosomal/lysosomal system. Recently the primary disease-causing gene, NPC1, was identified, but few clues regarding its potential function(s) could be derived from its predicted amino acid sequence. Therefore, efforts were directed at characterizing the subcellular location of the NPC1 protein. Initial studies with a FLAG-tagged NPC1 cDNA demonstrated that NPC1 is a glycoprotein that associates with the membranes of a population of cytoplasmic vesicles. Immunofluorescence microscopy using anti-NPC1 polyclonal antibodies confirmed this analysis. Double-label immunofluorescence microscopy and subcellular fractionation studies indicated that NPC1 associates predominantly with late endosomes (Rab9 GTPase-positive vesicles) and, to a lesser extent, with lysosomes and the trans-Golgi network. When cholesterol egress from lysosomes was blocked by treatment of cells with U18666A, the NPC1 location shifted from late endosomes to the trans-Golgi network and lysosomes. Subcellular fractionation of liver homogenates from U18666A-treated mice confirmed these observations. These data suggest that U18666A may inhibit the retrograde transport of NPC1 from lysosomes to late endosomes for subsequent transfer to the trans-Golgi network (Higgins, 1999).

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. Organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. It is concluded that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery (Ko, 2001).

The NPC1 protein is a multipass transmembrane protein whose deficiency causes the autosomal recessive lipid storage disorder Niemann-Pick type C1. NPC1 localizes predominantly to late endosomes and has a dileucine motif located within a small cytoplasmic tail thought to target the protein to this location. Previous data have suggested that the protein can reach its correct location in the absence of its cytoplasmic tail, suggesting that other signals contribute to NPC1 targeting. By using various FLAG-tagged and CD32-NPC1 chimeric fusion constructs, it was shown that multiple signals are responsible for the trafficking of NPC1 to the endosomal compartment, including the dileucine motif and a previously unidentified signal residing within the putative sterol-sensing domain transmembrane domain 3. Neither region alone was capable of directing heterologous CD32 fusions to late endosomes exclusively via the trans-Golgi network to the late endosome route taken by wild-type NPC1; transmembrane domain 3 was unable to maintain CD32 in late endosomes, indicating that two or more signals work in concert to target and retain NPC1 in this compartment. In addition it was confirmed that the tail dileucine motif is not essential for NPC1 targeting to late endosomes, and the implications of this finding were discussed along with the previously unappreciated role for transmembrane domain 3 in NPC1 localization and function (Scott, 2004).

Niemann-Pick type C and lysosomal transport

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)⁺/mannose 6-phosphate receptor^- vesicles that can be distinguished from cholesterol-enriched LAMP2⁺ lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21°C, consistent with vesicle-mediated transfer. These findings suggest that NPC1⁺ vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. It is concluded that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol (Neufeld, 1999).

Niemann-Pick type C lipid storage defect

Niemann-Pick disease type C (NPC) is a lethal neurologic storage disorder of children most often caused by a defect in the protein NPC1. To better understand the disease the cellular and morphological alterations occurring in murine, feline, and human NPC were thoroughly characterized. Using immunocytochemistry and filipin histochemistry it was shown that both gangliosides and unesterified cholesterol are differentially stored in neurons of the cerebral cortex, cerebellum, and hippocampus, as well as in liver. Double fluorescence labeling revealed that GM2 ganglioside and unesterified cholesterol were partially co-localized in vesicular structures, and triple fluorescence labeling utilizing a LAMP-1 antibody identified many of these organelles as part of the late endosomal/lysosomal pathway. These observations, coupled with the proposed role of NPC1 in intracellular cholesterol movement, suggest that GM3 and GM2 gangliosides as well as unesterified cholesterol may be retrogradely cleared from late endosomes/lysosomes by an NPC1-dependent mechanism. Cellular consequences of the NPC metabolic defect as shown by parvalbumin immunocytochemistry and rapid Golgi staining, respectively, revealed characteristic axonal spheroids on GABAergic neurons and ectopic dendritogenesis that followed a species-specific gradient of: mouse < feline < human. These studies suggest that the homeostatic regulation of gangliosides and cholesterol in neurons is mediated by NPC1 and that perturbations in this mechanism cause a complex neuronal storage disorder (Zervas, 2001a).

Niemann-Pick type C (NPC) disease is a cholesterol lipidosis caused by mutations in NPC1 and NPC2 gene loci. Most human cases are caused by defects in NPC1, as are the spontaneously occurring NPC diseases in mice and cats. NPC1 protein possesses a sterol-sensing domain and has been localized to vesicles that are believed to facilitate the recycling of unesterified cholesterol from late endosomes/lysosomes to the ER and Golgi. In addition to accumulating cholesterol, NPC1-deficient cells also accumulate gangliosides and other glycosphingolipids (GSLs), and neuropathological abnormalities in NPC disease closely resemble those seen in primary gangliosidoses. These findings led to a hypothesis that NPC1 may also function in GSL homeostasis. To evaluate this possibility, murine and feline NPC models were treated with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glucosylceramide synthase, a pivotal enzyme in the early GSL synthetic pathway. Treated animals showed delayed onset of neurological dysfunction, increased average life span (in mice), and reduced ganglioside accumulation and accompanying neuropathological changes. These results are consistent with the hypothesis and with GSLs being centrally involved in the pathogenesis of NPC disease, and they suggest that drugs inhibiting GSL synthesis could have a similar ameliorating effect on the human disorder (Zervas, 2001b).

Niemann-Pick type C (NPC) disease is a lysosomal disorder commonly caused by a recessive mutation in NPC1, which encodes an integral membrane protein with regions of homology to the morphogen receptor, Patched, and to 3-hydroxy-3-methylglutaryl coenzyme A reductase. Neurons in NPC disease exhibit extensive storage of free cholesterol and glycosphingolipids (GSLs), including GM2 and GM3 gangliosides. Most studies have viewed cholesterol storage as primary, with NPC1 functioning as a retroendocytic transporter for regulation of cholesterol homeostasis. This study analyzes the effects of genetically depriving NPC neurons of complex gangliosides by creating mice doubly deficient in both NPC1 and the GSL synthetic enzyme, GM2/GD2 synthase (GalNAcT). Ganglioside and cholesterol expression in neurons of NPC1^-/-/GalNAcT^+/+, NPC1^-/-/GalNAcT^-/-, NPC1^+/+/GalNAcT^-/-, and WT mice was examined in situ by immunocytochemical and histochemical methods. Neurons in double-deficient mice lacked intraneuronal GM2 accumulation as expected, but remarkably also exhibited absence or dramatic reduction in free cholesterol. Neurons storing cholesterol consistently showed GM3 accumulation but some GM3-positive neurons lacked cholesterol storage. These findings provide a compelling argument that cholesterol sequestration in NPC1-deficient neurons is ganglioside dependent and suggest that the function of NPC1 in these cells may be more closely linked to homeostatic control of GSLs than cholesterol (Gondre-Lewis, 2003).

Niemann-Pick type C (NP-C) disease is a fatal, autosomal recessive, childhood neurodegenerative disease. The NP-C mouse recapitulates the cholesterol and sphingolipid storage, onset of neurological deficits, histopathological lesions, Purkinje cell loss and early death typical of the most severe form of human NP-C. Neurosteroids, steroids made in the brain, affect neuronal growth and differentiation, and modulate neurotransmitter receptors. Disordered cholesterol trafficking might disrupt neurosteroidogenesis, thereby contributing to the NP-C phenotype. NP-C mouse brain contains substantially less neurosteroid than wild-type brain and has an age-related decrease in the ability to synthesize 5alpha-dihydroprogesterone and allopregnanolone. Immunohistochemical assessment confirms a decrease in expression of 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase, especially in cerebellum. Neonatal administration of allopregnanolone delays the onset of neurological symptoms, increases Purkinje and granule cell survival, reduces cortical GM2 and GM3 ganglioside accumulation and doubles the lifespan of NP-C mice. Earlier administration increases effectiveness of treatment. Decreased production of allopregnanolone apparently contributes to the pathology of NP-C; thus, neurosteroid treatment may be useful in ameliorating progression of the disease (Griffin, 2004).

Niemann-Pick type C (NPC) is an autosomal recessive lipid storage disorder characterized by lysosomal accumulation of cholesterol and gangliosides resulting from a defect in intracellular lipid trafficking. The NPC1 gene encodes a 1278-amino acid integral membrane protein involved in the sub-cellular trafficking of lipids. The exact biological function of NPC1 remains unclear. Recent evidence suggests that NPC1 is a eukaryotic member of the RND permease family of transport proteins, which when expressed in bacteria is capable of transporting fatty acids. The goal of this project was to assess the role of NPC1 in the transport of fatty acids in primary human fibroblasts using normal fibroblasts and fibroblasts from patients with three lysosomal storage diseases: NPC, mucolipidosis IV, and Sandhoff disease. If NPC1 is a fatty acid transporter, fatty acid accumulation should be found only in NPC fibroblasts. Three experimental approaches were used to assess the role of NPC1 as a fatty acid transporter: (1) the accumulation versus metabolism of low density lipoprotein-derived oleic acid was evaluated; (2) the amount of free fatty acid present after growth in lipoprotein-containing media was assessed; (3) the cellular accumulation of acriflavine, a fluorescent substrate for a number of resistance-nodulation-cell division permease transporters, was assessed. The results indicate that fatty acid flux through NPC1-deficient lysosomes is normal (Passeggio, 2005).

Impaired autophagy in the lipid-storage disorder niemann-pick type c1 disease

Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. This study shows defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-β-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. These data suggest that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease (Sarkar, 2013).

Niemann-Pick type C1 and stem cell self renewal

Neural stem cells (NSC) are capable of giving rise to neurons, glia and astrocytes. Although self renewal and differentiation in neural stem cells are regulated by many genes, among these Notch and Numb, little is known of the role of defective genes on the self renewal and differentiation of neural stem cell from developing brain. The Niemann-Pick type C1 (NPC1) disease is one of the neurodegenerative diseases, caused by a mutation of NPC1 gene that affects the function of NPC1 protein. The ability of NSC self renewal and differentiation was investigated using a model of Niemann-Pick type C1 (NPC1) disease. The NPC1 disorder significantly affected the self renewal ability of neural stem cells, as well as the differentiation. Neural stem cell from NPC1^-/- mice showed impaired self renewal ability when compared to the NPC1^+/+ mice. These alterations were accompanied by the enhanced activity of p38 MAP kinases. Further, the specific p38 MAP kinase inhibitor, SB202190 improved the self renewal ability of NSCs from NPC^-/- mice. This indicates that the NPC1 deficiency can lead to lack of self renewal and altered differentiation of neural stem cells mediated by the activation of p38 MAP kinase impairing the generation of neurospheres from NPC1^-/-. Thus the NPC1 gene may play a crucial role in NSC self-renewal associated with p38 MAP kinase (Yang, 2005).

Cell death in Niemann-Pick type C disease

Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). This study investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death (Ko, 2005).

Correction of membrane traffic in NPC cells by Rab9 overexpression

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. Protein transduction has been uded to further investigate Rab9-mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22-Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 microM. A fraction of the VP22-Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22-Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY-lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL-receptor levels. In addition, reduction of intracellular cholesterol by VP22-Rab9 transduction was demonstrated in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease (Narita, 2005).

Identification and charactization of Niemann-Pick type C2 gene and protein

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. The disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol (Naureckiene, 2000).

Niemann-Pick type C1 (NPC1) disease is caused by defects in the NPC1 protein, which result in perturbation of subcellular cholesterol transport. To identify related proteins that may be involved in subcellular cholesterol trafficking, the expressed sequence tag (EST) database was searched to find homologues of human NPC1. A short, weakly similar EST was identified and used to obtain a full-length human cDNA of about 5 kb and two alternatively spliced transcripts. The gene, named NPC1L1, was mapped to chromosome 7p13, contained 20 exons, including an unusually large 1526-bp exon 2, and spanned approximately 29 kb. In contrast to NPC1, the NPC1L1 putative promoter region contained a sterol-regulatory element. The predicted protein shared 42% identity and 51% similarity with NPC1. Interestingly, NPC1L1 contains the conserved amino-terminal 'NPC1 domain' and the putative sterol-sensing domain, providing strong evidence that it is related to human NPC1 and suggesting that these may comprise a new family of NPC1-related proteins. However, the two differ with respect to their putative intracellular targeting signals. Collectively, these data suggest that NPC1L1 and NPC1 form part of a family of related proteins that may have similar functions at different subcellular locations, perhaps at sequential steps of the same cholesterol transport pathway (Davies, 2000b).

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice have substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice are completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding results in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency results in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia (Altmann, 2004).

NPC1L1, a recently identified relative of Niemann-Pick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 is highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest expression found in human liver and significant expression in the small intestine. In contrast, liver expression in mouse is extremely low with mouse small intestine exhibiting the highest NPC1L1 expression. A mouse knock-out model of NPC1L1 was generated and revealed that mice lacking a functional NPC1L1 have multiple lipid transport defects. Surprisingly, lack of NPC1L1 exerts a protective effect against diet-induced hyperlipidemia. Further characterization of cell lines generated from wild-type and knock-out mice revealed that in contrast to wild-type cells, NPC1L1 cells exhibit aberrant plasma membrane uptake and subsequent transport of various lipids, including cholesterol and sphingolipids. Furthermore, lack of NPC1L1 activity causes a deregulation of caveolin transport and localization, suggesting that the observed lipid transport defects may be the indirect result of an inability of NPC1L1 null cells to properly target and/or regulate caveolin expression (Davies, 2005).

Interaction between NPC1 and NPC2

NPC (Niemann-Pick type C) disease is a rare lipidosis characterized by the accumulation of LDL (low-density lipoprotein)-derived non-esterified cholesterol in the E/L (endosomal/lysosomal) system. The gene products that are responsible for the two NPC complementation groups are distinct and dissimilar, yet their cellular and disease phenotypes are virtually indistinguishable. To investigate the relationship between NPC1 and NPC2 and their potential role in NPC disease pathogenesis, a method was developed for the rapid and efficient isolation of late endocytic vesicles from mouse liver by magnetic chromatography. Late endosomes from Wt (wild-type) and NPC1 mice were found to differ not only in their cholesterol and sphingomyelin content, as expected, but also in their non-esterified ('free') fatty acid content, with NPC1 vesicles showing an approx. 7-fold increase in non-esterified fatty acid levels compared with Wt vesicles. Furthermore, the NPC2 protein is in an incompletely deglycosylated form in NPC1 late endosomes by a mechanism that is specific to the NPC2 protein and not a global aberration of protein glycosylation/deglycosylation or trafficking, since NPC2 secreted from NPC1 cells is indistinguishable from that secreted from Wt cells. Also, a greater proportion of the normally soluble cellular NPC2 protein partitions with detergent-insoluble late endosomal internal membrane domains in NPC1 vesicles. Although a small amount of the NPC2 protein associates with these membranes in Wt vesicles, this localization becomes much more pronounced in NPC1 vesicles. These results suggest that the function of the NPC2 protein may be compromised as well in NPC1 endosomes, which might explain the paradoxical phenotypic similarities of the two NPC disease complementation groups (Chen, 2005).

REFERENCES

Search PubMed for articles about Drosophila Niemann-Pick Type C-1a

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date revised: 25 July 2022

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