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).

GENE STRUCTURE

cDNA clone length - 4191 bp

Bases in 5' UTR - 230

Exons - 12

Bases in 3' UTR - 97

PROTEIN STRUCTURE

Amino Acids - 1287

Structural Domains

Like Drosophila, vertebrate genomes encode a pair of closely related NPC1 homologs. The more recently identified vertebrate protein, designated NPC1L1, has been found to function in sterol absorption in the intestine (Altmann, 2004; Davies, 2000b). To explore the possibility that the Drosophila NPC1a and NPC1b proteins are orthologous to specific vertebrate NPC1 family members the sequences of the human NPC1 family members were compared to the two Drosophila homologs. Results of this analysis indicate that the Drosophila NPC1a and NPC1b proteins are both more similar to human NPC1 and to each other (38% identity) than they are to NPC1L1. NPC1a displays greater sequence similarity to both NPC1 and NPC1L1 than NPC1b does, raising the possibility that the gene duplication events that gave rise to multiple NPC1 family members in flies and humans occurred independently in these organisms. While these observations complicate efforts to define strict orthologous relationships within this gene family, several sequence features suggest that NPC1a and NPC1b may function equivalently to NPC1 and NPC1L1, respectively. In particular, the findings that NPC1a has greater sequence similarity to NPC1 and that NPC1a and NPC1 (but not NPC1b or NPC1L1) share a C-terminal lysosomal targeting motif suggest that NPC1a may play a functional role that more closely parallels that of NPC1 (Ioannou, 2000; Scott, 2004). Furthermore, the NPC1L1 and NPC1b polypeptides share a putative YQRL Golgi retention signal and no such sequence is found in NPC1 or NPC1a (Fluegel, 2005)

A BLAST search of the Drosophila melanogaster genome sequence identified two NPC1-like genes that have been named dnpc1a and dnpc1b (Flybase term: NPC1b). dnpc1a is predicted to encode a 1287 amino acid protein with 44% similarity and 63% identity to human NPC1, and 38% identity and 56% similarity to human NPC1L1. dNPC1b is a 1254 amino acid protein that shares 38% sequence identity and 55% similarity to human NPC1, and 33% identity and 51% similarity to human NPC1L1. The dNPC1a protein sequence is 39% identical and 58% similar to the dNPC1b sequence (Huang, 2005).

date revised: 26 January 2006 Niemann-Pick Type C-1 : Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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