The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. This study used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. A conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter was identified using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development (Xiang, 2010).
Ecdysone hormone produced in the ring gland plays a central role in regulating insect development. This study identified RSE, a ring gland-specific cis-regulatory element, in the promoter of the ecdysone biosynthesis-related gene npc1. In addition, br, an ecdysone early response gene, was found to be a key regulator for the ring gland expression of npc1. Moreover, br seems to regulate the expression of many other ecdysone biosynthesis-related genes in the ring gland (Xiang, 2010).
The functions of br have been studied extensively. As an ecdysone early response gene, it regulates the transcription of many late response genes, including L71, sgs-4, and hsp23, in response to the ecdysone signal. br also regulates the expression of Drosophila caspase Dronc, which is important for programmed cell death during metamorphosis. The null allele of br, brnpr-3 displays an ecdysone-deficient phenotype, arresting at wandering third instar for a long time, which is consistent with its role in mediating the ecdysone response. However, it was found that implanting a wild-type ring gland can partially rescue the brnpr-3 phenotype, implying that br mutants may be partial ecdysone deficient. Previous studies on the role of BR have mainly focused on ecdysone-responding tissues, for instance the salivary gland. It remains unclear whether BR also affects ecdysone biosynthesis. The current findings suggest that besides having a role in triggering ecdysone late response gene expression in larval peripheral tissues, br also has an important role in regulating ecdysone biosynthesis in the ecdysone-producing organ. In addition, the fact that the larval-arrest phenotype in the animals with ring gland-specific knockdown of br was only partially rescued by ecdysone indicates that br may have other roles in the ring gland. In agreement with that, experiments with ring gland-specific overexpression of different br isoforms revealed that br may function in regulating the degeneration of ring gland (Xiang, 2010).
This study implies the existence of a positive feedback loop in which ecdysone regulates the transcriptional expression of the early response gene br and br could subsequently augment the transcription of ecdysone biosynthesis-related genes to further boost ecdysone production in the ring gland. The feedback regulation of ecdysone biosynthesis has been well documented in insects and ecdysone could have both positive and negative roles on ecdysone biosynthesis. What is the significance of such a positive feedback regulation mechanism? During development, ecdysone levels increase and decrease rapidly before molt and after molt, respectively, as well as before pupariation and after pupariation. The mechanism by which such quick changes of ecdysone levels are achieved remains elusive. It is believed that the positive feedback regulation may facilitate the rapid increase in ecdysone biosynthesis. In contrast, this feedback may be involved in the subsequent decline in ecdysone biosynthesis after molt or pupariation. Therefore, this feedback regulation could help to fine-tune ecdysone biosynthesis within a small time window during rapid development (Xiang, 2010).
The initial aim of this study was to identify a ring gland-specific factor that acts on the RSE to regulate npc1 tissue-specific gene expression. However, several known ring gland-specific transcription regulators seem not to be required for npc1 expression. In contrast, BR, which is not a ring gland-specific protein, was proved to be vital for npc1 ring gland expression. These studies suggest that there are unknown factor(s) involved in npc1 transcriptional regulation. Wild-type nuclear extracts led to a high-molecular-weight shift of the RSE core probe, suggesting that these factors may form a large protein complex. Unfortunately, the identity of these unknown factors remains to be determined. More work, for example purifying BR interacting proteins to reveal their identity and to examine whether they are ring gland-specifically expressed, needs to be done (Xiang, 2010).
The RSE identified in this study is the first ring gland-specific element to be discovered. It is conserved through evolution in several Drosophila species. RSEs of other Drosophila species are active in D. melanogaster. Consistently, the regulator BR is also conserved. Moreover, the RSE core BR-Z4 binding site is present in a set of ecdysone biosynthesis genes, suggesting that the RSE is important for ecdysone biosynthesis. In addition, many ring gland-specific Gal4 lines have previously been reported, including 2-286, P0206, May60, and phm. Besides phm-Gal4, the regulatory sequences for these Gal4 lines are unknown. This study found that while the activity of npc1-Gal4 is regulated by br, the activity of P0206-Gal4 is also regulated by br. In contrast, the RSE is likely not the only ring gland-specific element. There are other ring gland-specific transcription regulators, such as ecd, mld, and woc, which do not act on the RSE of npc1. The targeting regulatory elements for these genes have not yet been identified. Finding such elements would undoubtedly advance knowledge of the regulation of ecdysone biosynthesis (Xiang, 2010).
As an evolutionary conserved gene, npc1 is important for cholesterol trafficking in many other systems including mammals. In mice, npc1 is vital for neurosteroid biosynthesis, which is likely a key factor determining the neurodegenerative phenotype of npc1 mutants. These studies on the regulation of Drosophila npc1 tissue-specific expression by br may contribute to studies on the regulation of neurosteroid biosynthesis in higher animals (Xiang, 2010).
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, 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 NPC1a57A 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).
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).
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).
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).
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date revised: 15 October 2008
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