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).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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).

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).

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