|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 5 September 2007
doi: 10.1242/dev.004572
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Departments of Developmental Biology, Genetics and Bioengineering, Howard
Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA
94305-5439, USA.
2 Laboratory of Molecular and Developmental Biology, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beijing, 100101,
China.
3 Department of Biology, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-3280, USA.
4 Department of Molecular and Cellular Physiology, Stanford University School of
Medicine, Stanford, CA 94305-5439, USA.
* Author for correspondence (e-mail: mscott{at}stanford.edu)
Accepted 18 July 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Niemann-Pick type C, Sterol, Ecdysteroid, Drosophila
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). 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.
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
(Patterson, 2003). 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
(Liscum and Faust, 1987). NPC
disease is a progressive neurodegenerative disorder in which the degeneration
of cerebellar Purkinje neurons is most prominent
(Higashi et al., 1993).
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 (Griffin et al.,
2004; Langmade et al.,
2006).
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
(Patterson, 2003). The large
Npc1 protein has 13 transmembrane domains and a sterol-sensing domain (SSD)
(Carstea et al., 1997;
Loftus et al., 1997). 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 (Naureckiene et al.,
2000). 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 (Friedland et al.,
2003; Ko et al.,
2003). Npc2 may serve as a lysosomal cholesterol transporter,
rapidly transporting cholesterol to acceptor membranes
(Cheruku et al., 2006).
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.
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 (Berger et al.,
2005; Higaki et al.,
2004; Li et al.,
2004; Malathi et al.,
2004). We and the L. Pallanck laboratory have previously created
Drosophila NPC models using npc1a (also referred to as
NPC1 - FlyBase) mutations (Fluegel
et al., 2006; Huang et al.,
2005). 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
(Clark and Block, 1959). 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
(Thummel, 1996).
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.
Based on the findings in npc1 mutant worms, flies and mice, we
proposed a cholesterol shortage model of NPC disease
(Huang et al., 2005). 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. In the studies reported here, we examined the functions
of Npc2 proteins in Drosophila. Our results further support the
cholesterol shortage model proposed previously.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Three alleles of npc2a (npc2a239,
npc2a271 and npc2a376) and three alleles
of npc2b (npc2b18, npc2b19 and
npc2b22) were isolated. All mutants were back-crossed to
wild-type (Canton-S) flies three times before further phenotypic
characterizations. The molecular lesions were determined using genomic DNA
polymerase chain reactions and by sequencing. The coding regions of
npc2a and npc2b are entirely deleted by the mutations in
their corresponding sets of alleles.
Molecular biology
Eight npc2-like genes (npc2a-h) were found by BLAST
searches of the Drosophila melanogaster genome with the sequence of
the human NPC2 protein. The cDNAs corresponding to the npc2 gene
family were amplified by RT-PCR and sequenced. The protein sequences were then
deduced from the cDNA sequences. The gene structures of all but one
(npc2e) of the npc2 genes were predicted correctly in
FlyBase. npc2e (CG31410) has an extra intron compared to the FlyBase
prediction and the correct coding sequence aligns well with other Npc2
proteins. The UAST-npc2a and UAST-npc2b constructs were made
by inserting full-length cDNAs into the EcoRI site of the pUAST
vector.
Ecdysteroid titer measurement
First-instar wild-type and mutant larvae were collected into 1.5 ml tubes
(100 larvae/tube) and kept at -80°C until assayed for whole body
ecdysteroid content. Larvae were homogenized by sonication (Sonic
Dismembrator, Fisher) and extracted exhaustively with both methanol and
ethanol. Pooled solvents for each replicate of 100 larvae were evaporated
under low pressure into 2 ml plastic tubes and the dried residue subjected to
RIA employing the H22 antiserum (Warren et
al., 1988). For each genotype, 600-800 larvae were used.
Sterol and 20E feeding
For npc2a; npc2b double mutants, each group of 200 first-instar
larvae was placed on apple juice plates with baker's yeast paste containing
supplementary sterols, as described previously
(Huang et al., 2005), and the
lethal phases were determined by larval spiracle and mouth hook development.
The final concentrations for the supplements used were: cholesterol, 0.14
mg/g; 7-dehydrocholesterol, 0.14 mg/g; and 20E, 8 µg/g.
Sterol quantitation
The sterol content in larvae was quantified by following a published
protocol (Fluegel et al.,
2006). The Amplex Red cholesterol assay kit (Molecular Probes) was
used to assess sterol content in wandering third-instar animals. Larvae were
collected and washed before being weighed and homogenized in 150 mM NaCl, 2 mM
EGTA, 50 mM Tris pH 7.5, to make a 100 mg/ml larval homogenate. The homogenate
was spun at 5000 rpm for 5 minutes to pellet cuticle debris, and the
supernatant was used for sterol content assays. Fluorescence was measured with
a fluorescence spectrophotometer with a 560/585 nm filter set.
Filipin staining and immunohistology
For filipin staining of free sterols, tissues were fixed in 4%
paraformaldehyde for 30 minutes, washed twice in PBS and stained with 50
µg/ml filipin (Sigma) PBS solution for 30 minutes. Samples were then washed
twice with PBS before mounting them in Vectashield mounting medium. For TUNEL
analysis, aged brains were dissected and fixed (PBS, 4% paraformaldehyde) for
20 minutes at room temperature. Tissues were washed twice in PBS, once in
H2O plus 0.1% Triton X-100 and 0.1% sodium citrate for 10 minutes,
and then twice in PBS. TUNEL analysis was performed by following the
manufacturer's instructions (Boehringer Mannheim). TUNEL and neuron double
labeling was performed using an antibody against the pan-neuronal marker Elav
(Iowa Hybridoma Bank). Synaptotagmin staining (anti-Synaptotagmin, from Dr
Hugo Bellen) was performed using standard techniques
(Littleton et al., 1993). All
images were collected using a compound microscope and a cold CCD camera.
Life span analysis
For each genotype, 10 vials containing a total of 200 flies were passed
into fresh vials every 4 days, at which time the number of dead flies was
recorded.
Methods for in situ hybridization to detect mRNA in overnight embryo
collections and for electron microscopy were described previously
(Huang et al., 2005).
Malpighian tubules from third-instar larvae were dissected first, then fixed
in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH
7.4, followed by further processing for electron microscopy.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Of the eight Npc2-like proteins, Npc2a (also referred to as NPC2 - FlyBase) has the highest sequence identity (36%) and similarity (53%) to human NPC2 (Fig. 1B). 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 (Fig. 1A). Both groups of genes presumably arose from gene duplication events.
Crystal structure determination and mutational analyses have shown that
Npc2 has three disulfide bonds and forms a hydrophobic core implicated in
cholesterol binding (Friedland et al.,
2003; Ko et al.,
2003). 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 (Ko et al., 2003). 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)
(Fig. 1B). 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)
(Fig. 1B). 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 (Fig.
1B) (Berger et al.,
2005).
|
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 (position 1 in Fig. 1B). Two others, npc2c and e, have two introns in the same positions (positions 1 and 2 in Fig. 1B). The eighth gene, npc2f, has three introns (positions 1, 2 and 3 in Fig. 1B). Interestingly, the intron positions (positions 1, 2 and 3 in Fig. 1B) in the D. melanogaster 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 (position 4 in Fig. 1B). 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.
Pattern of npc2a-h transcription in time and space
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 (Fig. 2).
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 (Fig. 2A-D).
npc2b is expressed at the highest levels in the trachea and
hypopharynx (Fig. 2I).
npc2g is specifically expressed in head mesoderm and fat body
(Fig. 2G,H). npc2d and
npc2h transcripts could be detected only in salivary gland
(Fig. 2F), while npc2e
is expressed in hindgut (not shown). The expression of npc2c and
npc2f was not detected by in situ hybridization at any time during
embryogenesis.
As 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. We could detect moderate levels of gene expression in larval ring glands, brains and imaginal discs for several npc2 genes, including npc2a and npc2b (Fig. 2K,L and data not shown).
Npc2a is required for sterol homeostasis
Because npc2a has the broadest expression pattern among the eight
genes studied, and the highest protein sequence similarity to vertebrate Npc2,
we focused initially on characterizing npc2a function using mutant
phenotypic analysis. Through P element imprecise excision we generated three
deletion alleles (npc2a239, npc2a271
and npc2a376; Fig.
3A). 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 (Fig.
2E).
|
). 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 (Fig. 3B-G). Most tissues had many such spots of accumulated sterol, except trachea, where we found fewer puncta. 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 (see below), indicating that this phenotype is indeed due to npc2a mutation.
We further examined the structure of mutant npc2a/npc2a cells using electron microscopy. Large multi-lamellar body and multi-vesicular body structures were found in npc2a mutant Malpighian tubules (Fig. 4), 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 (Fig. 4B and C, respectively). 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.
Ecdysteroid deficiency in npc1a but not npc2a mutants
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? We have suggested previously that
the first-instar larval lethality of npc1a is due to ecdysteroid
deficiency, although this was inferred rather than measured directly
(Huang et al., 2005). The
difference in phenotypes between npc1a and npc2a homozygotes
could reflect different ecdysteroid levels.
We have now directly measured ecdysteroid levels 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)
(Garen et al., 1977;
Kraminsky et al., 1980;
Neubueser et al., 2005). 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 our previous hypothesis that the
first-instar lethality of npc1a/npc1a mutants is due to ecdysteroid
deficiency.
Redundant roles of npc2a and npc2b in sterol homeostasis and ecdysteroid biosynthesis
The ecdysteroid titer results do not explain why apparently similar defects
in sterol distribution are associated with a shortage of sterol substrate for
ecdysteroid biosynthesis in npc1a but not npc2a mutants.
There are at least two possibilities. First, npc1a and npc2a
may function differently in ecdysteroidogenesis, so that only npc1a
but not npc2a is involved in sterol transport to the mitochondria.
This could be true despite the apparently similar overall accumulation of
sterol in filipin-stained compartments. Alternatively, the difference could be
due to redundant functions of the multiple npc2 genes. Perhaps in the
npc2a/npc2a mutants a substantial amount of sterol reaches the
mitochondria, transported by other Npc2 family protein-mediated processes.
|
To determine whether sterol accumulation in npc2a mutants is prevented by npc2b, we generated three npc2b deletion alleles (npc2b18, npc2b19 and npc2b22) of npc2b by imprecise P element excision. The whole coding region of npc2b was completely deleted in these three alleles. In homozygous npc2b mutant animals, no in situ hybridization signal could be detected with an npc2b antisense probe, so as expected the new alleles of npc2b were nulls (Fig. 2). Like npc2a homozygotes, npc2b homozygotes and flies carrying an npc2b allele in trans to a genetic deficiency were viable and fertile.
No sterol accumulation was observed in any npc2b mutant tissues, including the trachea, where we know the gene is preferentially transcribed (Fig. 5B). However, npc2a/npc2a; npc2b/npc2b doubly homozygous mutants had a large number of filipin-stained puncta in the trachea. The level of sterol particles was similar to sterol accumulation in npc1a/npc1a mutants (Fig. 5D). We conclude that npc2a and npc2b function redundantly in sterol trafficking, at least in trachea.
Although both single mutants were viable, fertile and were not developmentally delayed, npc2a; npc2b double mutants died as larvae or pupae and the third-larval instar was prolonged. Aside from a small percentage of animals (about 17%) that died in the first or second larval stage, the majority of npc2a/npc2a; npc2b/npc2b double mutants molted to the third instar quite normally. They remained in the third instar for 3-6 days, compared with about 2 days for wild-type animals. Twenty-six percent died while still in the third-instar stage, while the remaining 57% formed pupae (Fig. 6A). About a tenth of the mutant pupae developed to the adult stage, but they were sick and usually died within 2 weeks (Fig. 6A). For this reason we were not able to establish homozygous double mutant stocks.
Most of the npc2a/npc2a; npc2b/npc2b double mutants could be rescued by feeding them a diet enriched with cholesterol, 7-dehydrocholesterol or 20E (Fig. 6A). The prolonged third instar of the double mutants, together with the results of the rescue experiment, suggests that the ecdysteroid level is relatively low in the double mutants. In the presence of sufficient substrate, npc2a/npc2a; npc2b/npc2b mutants were evidently able to synthesize enough ecdysteroid for fairly normal development.
As with npc1a mutants, the insufficiency of sterol substrate appeared to be the main problem for npc2a/npc2a; npc2b/npc2b double mutants. The similarity of the double npc2 homozygotes to npc1a homozygotes suggests that npc1a has irreplaceable functions, while the two npc2 genes tested to date have somewhat redundant functions. Both Npc1 and Npc2 are necessary to regulate sterol homeostasis and carry out adequate biosynthesis of 20E.
Tissue-specific requirement of npc2
Our results agree well with the hypothesis that Npc1 and Npc2 promote
efficient intracellular sterol trafficking for ecdysone biosynthesis. To
further pinpoint the roles of Npc2, we examined the sterol level in
npc2 mutants and possible tissue-specific requirements for
npc2. Despite the altered filipin staining patterns in many tissues,
the overall level of sterol was not much different in Drosophila
npc1a mutants compared to controls
(Fluegel et al., 2006). We
measured sterol levels in npc2 mutants and found a similar result:
the overall level of sterol was not significantly changed in
npc2a/npc2a or npc2b/npc2b single mutants or in
npc2a/npc2a;npc2b/npc2b double mutants
(Fig. 6B).
|
Expression of UAST-npc2a or UAST-npc2b only in the ring gland, using the 2-286-Gal4 driver, rescued the lethality of the double mutant: 78% of the double mutants survived to adulthood in the presence of 2-286-Gal4>UAST-npc2a and 86% survived to adulthood in the presence of 2-286-Gal4>UAST-npc2b. These findings are consistent with the conclusion that a defect in ecdysone biosynthesis is the main cause of the larval lethal phenotype. By contrast, pan-neuronal expression of UAST-npc2a or UAST-npc2b did not show any rescuing activity.
Neuronal phenotypes of npc2 mutants
In addition to cellular defects in cholesterol homeostasis, mammalian NPC
mutants have neuronal and behavioral defects, including region-specific
neurodegeneration, ataxia, dementia and early death. We examined
Drosophila npc2 mutants in detail to search for potential neuronal
phenotypes.
Drosophila neurodegenerative mutants are often associated with a
short life span and numerous large vacuoles in the brain
(Min and Benzer, 1999;
Palladino et al., 2002). We
assessed the adult life span of npc2a mutants. npc2a mutants
displayed a slightly shorter life span compared with wild type
(Fig. 7A). For example, by day
52 more than 60% of the wild-type flies were still alive compared with fewer
than 10% of the npc2a/npc2a mutants. Fifty percent of the
npc2a mutants died by day 36, a time when more than 90% of the
wild-type flies remained alive.
We sectioned adult brains from 30-day-old npc2a/npc2a and wild-type animals to look for the presence of large vacuoles indicative of neurodegeneration. We found no evidence of any neurodegenerative vacuoles (data not shown). Reasoning that subtle neurodegeneration may not cause the formation of large vacuoles, we next used TUNEL staining to look for apoptotic cells in adult brains. Compared with wild type, we found few TUNEL-positive cells in 30-day-old npc2a/npc2a mutant brains (Fig. 7B). By contrast, many TUNEL-positive cells were present in 7-day-old npc2a/npc2a; npc2b/npc2b double homozygous brains and in tracheal cells that extended along the top of the brains (Fig. 7B). We double-stained mutant flies with antibodies against the pan-neuronal marker Elav and for TUNEL-positive cells. Most of the TUNEL-positive cells were neurons (Fig. 7C). Similar TUNEL-positive cells, indicative of neurodegeneration, were found in Drosophila npc1a mutants (data not shown). Thus Drosophila npc1/2 mutants faithfully display cholesterol accumulation and neurodegenerative phenotypes analogous to those of mammalian NPC mutants.
Mammalian Npc1 has been found in axons as well as presynaptic nerve
terminals, and Npc1/Npc1 mutant mice have mild morphological changes
in presynaptic nerve terminal (Karten et
al., 2006). For this reason, Synaptotagmin staining of
third-instar larvae was performed to examine neuromuscular junction (NMJ)
structure and axon morphology. We found no difference in NMJ morphology in
npc2a/npc2a mutants, but axonal transport defects were detected at a
low frequency (two to three sites per animal). These defects took the form of
accumulated Synaptotagmin within axon tracts
(Fig. 7C). The significance of
this phenotype for neural function remains to be learned.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
|
). The trafficking defect may occur earlier than sterol
accumulation and compartmentalization in the diseased state as well. The
neurodegeneration could then be a consequence of either sterol accumulation or
of other outcomes of defective trafficking. Evidence in favor of the latter
idea comes from monitoring the degeneration of cerebellar Purkinje neurons in
Npc1/Npc1 mutant mice (Ko et al.,
2005). The Purkinje cells that die are not those that have the
highest cholesterol accumulation. Other outcomes of defective trafficking may
therefore kill neurons, such as a failure to transport sterol substrate to
ER/mitochondria for steroid hormone synthesis, as we have suggested in a model
of proposed cholesterol shortage (Huang et
al., 2005).
|
Redundancy of Npc2 proteins in Drosophila
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?
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. Our 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). As
insects are cholesterol auxotrophs and need external sterol sources for growth
(Clark and Block, 1959), 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, as 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. As 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.
|
). 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
(Akaboshi and Ohno, 1995;
Somers et al., 2001). 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.
Our studies of Drosophila npc2 genes are consistent with the
sterol-shortage model proposed previously
(Huang et al., 2005). 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 (Tsutsui et al.,
1999). Npc1/Npc1 mutant mice are deficient in
neurosteroids, and administration of supplementary allopregnanolone reduces
the symptoms of NPC disease (Griffin et
al., 2004). 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.
npc1a and npc2 define a new kind of gene involved in 20E biosynthesis
Our 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 (Gilbert et al.,
2002). 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
(Gilbert et al., 2002). The
larval arrest phenotypes resulting from ablating Drosophila neurons
that produce PTTH are consistent with a role in governing ecdysteroid
biosynthesis (X.H. and M.P.S., unpublished). 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
(Caldwell et al., 2005). The
third class of genes includes nuclear transcription factors and regulators,
such as ftz-f1, ecd, woc and mld
(Gaziova et al., 2004;
Neubueser et al., 2005;
Parvy et al., 2005;
Wismar et al., 2000). 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
(Chavez et al., 2000;
Gilbert and Warren, 2005;
Ono et al., 2006;
Petryk et al., 2003;
Warren et al., 2002).
The present study, together with our previous study on 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 our 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 (Stocco,
2001).
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Akaboshi, S. and Ohno, K. (1995). [Niemann-Pick
disease type C]. Nippon Rinsho
53,3036
-3040.[Medline]
Berger, A. C., Vanderford, T. H., Gernert, K. M., Nichols, J.
W., Faundez, V. and Corbett, A. H. (2005). Saccharomyces
cerevisiae Npc2p is a functionally conserved homologue of the human
Niemann-Pick disease type C 2 protein, hNPC2. Eukaryotic
Cell 4,1851
-1862.
Caldwell, P. E., Walkiewicz, M. and Stern, M.
(2005). Ras activity in the Drosophila prothoracic gland
regulates body size and developmental rate via ecdysone release.
Curr. Biol. 15,1785
-1795.[CrossRef][Medline]
Carstea, E. D., Morris, J. A., Coleman, K. G., Loftus, S. K.,
Zhang, D., Cummings, C., Gu, J., Rosenfeld, M. A., Pavan, W. J., Krizman, D.
B. et al. (1997). Niemann-Pick C1 disease gene: homology to
mediators of cholesterol homeostasis. Science
277,228
-231.
Chavez, V. M., Marques, G., Delbecque, J. P., Kobayashi, K.,
Hollingsworth, M., Burr, J., Natzle, J. E. and O'Connor, M. B.
(2000). The Drosophila disembodied gene controls late embryonic
morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic
ecdysone levels. Development
127,4115
-4126.[Abstract]
Cheruku, S. R., Xu, Z., Dutia, R., Lobel, P. and Storch, J.
(2006). Mechanism of cholesterol transfer from the Niemann-Pick
type C2 protein to model membranes supports a role in lysosomal cholesterol
transport. J. Biol. Chem.
281,31594
-31604.
Clark, A. J. and Block, K. (1959). The absence
of sterol synthesis in insects. J. Biol. Chem.
234,2578
-2582.
Farese, R. V., Jr and Herz, J. (1998).
Cholesterol metabolism and embryogenesis. Trends
Genet. 14,115
-120.[CrossRef][Medline]
Fluegel, M. L., Parker, T. J. and Pallanck, L. J.
(2006). Mutations of a Drosophila NPC1 gene confer sterol and
ecdysone metabolic defects. Genetics
172,185
-196.
Friedland, N., Liou, H. L., Lobel, P. and Stock, A. M.
(2003). Structure of a cholesterol-binding protein deficient in
Niemann-Pick type C2 disease. Proc. Natl. Acad. Sci.
USA 100,2512
-2517.
Garen, A., Kauvar, L. and Lepesant, J. A.
(1977). Roles of ecdysone in Drosophila development.
Proc. Natl. Acad. Sci. USA
74,5099
-5103.
Gaziova, I., Bonnette, P. C., Henrich, V. C. and Jindra, M.
(2004). Cell-autonomous roles of the ecdysoneless gene in
Drosophila development and oogenesis. Development
131,2715
-2725.
Gilbert, L. I. and Warren, J. T. (2005). A
molecular genetic approach to the biosynthesis of the insect steroid molting
hormone. Vitam. Horm.
73, 31-57.[Medline]
Gilbert, L. I., Rybczynski, R. and Warren, J. T.
(2002). Control and biochemical nature of the ecdysteroidogenic
pathway. Annu. Rev. Entomol.
47,883
-916.[CrossRef][Medline]
Griffin, L. D., Gong, W., Verot, L. and Mellon, S. H.
(2004). Niemann-Pick type C disease involves disrupted
neurosteroidogenesis and responds to allopregnanolone. Nat.
Med. 10,704
-711.[CrossRef][Medline]
Higaki, K., Almanzar-Paramio, D. and Sturley, S. L.
(2004). Metazoan and microbial models of Niemann-Pick Type C
disease. Biochim. Biophys. Acta
1685,38
-47.[Medline]
Higashi, Y., Murayama, S., Pentchev, P. G. and Suzuki, K.
(1993). Cerebellar degeneration in the Niemann-Pick type C mouse.
Acta Neuropathol. 85,175
-184.[Medline]
Huang, X., Suyama, K., Buchanan, J., Zhu, A. J. and Scott, M.
P. (2005). A Drosophila model of the Niemann-Pick type C
lysosome storage disease: dnpc1a is required for molting and sterol
homeostasis. Development
132,5115
-5124.
Karten, B., Campenot, R. B., Vance, D. E. and Vance, J. E.
(2006). The Niemann-Pick C1 protein in recycling endosomes of
presynaptic nerve terminals. J. Lipid Res.
47,504
-514.
Ko, D. C., Gordon, M. D., Jin, J. Y. and Scott, M. P.
(2001). Dynamic movements of organelles containing Niemann-Pick
C1 protein: NPC1 involvement in late endocytic events. Mol. Biol.
Cell 12,601
-614.
Ko, D. C., Binkley, J., Sidow, A. and Scott, M. P.
(2003). The integrity of a cholesterol-binding pocket in
Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels.
Proc. Natl. Acad. Sci. USA
100,2518
-2525.
Ko, D. C., Milenkovic, L., Beier, S. M., Manuel, H., Buchanan,
J. and Scott, M. P. (2005). Cell-autonomous death of
cerebellar purkinje neurons with autophagy in niemann-pick type C disease.
PLoS Genet. 1,e7
.[CrossRef]
Kraminsky, G. P., Clark, W. C., Estelle, M. A., Gietz, R. D.,
Sage, B. A., O'Connor, J. D. and Hodgetts, R. B. (1980).
Induction of translatable mRNA for dopa decarboxylase in Drosophila: an early
response to ecdysterone. Proc. Natl. Acad. Sci. USA
77,4175
-4179.
Langmade, S. J., Gale, S. E., Frolov, A., Mohri, I., Suzuki, K.,
Mellon, S. H., Walkley, S. U., Covey, D. F., Schaffer, J. E. and Ory, D.
S. (2006). Pregnane X receptor (PXR) activation: a mechanism
for neuroprotection in a mouse model of Niemann-Pick C disease.
Proc. Natl. Acad. Sci. USA
103,13807
-13812.
Li, J., Brown, G., Ailion, M., Lee, S. and Thomas, J. H.
(2004). NCR-1 and NCR-2, the C. elegans homologs of the human
Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer
formation pathways. Development
131,5741
-5752.
Liscum, L. and Faust, J. R. (1987). Low density
lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake
is defective in Niemann-Pick type C fibroblasts. J. Biol.
Chem. 262,17002
-17008.
Littleton, J. T., Bellen, H. J. and Perin, M. S.
(1993). Expression of synaptotagmin in Drosophila reveals
transport and localization of synaptic vesicles to the synapse.
Development 118,1077
-1088.[Abstract]
Loftus, S. K., Morris, J. A., Carstea, E. D., Gu, J. Z.,
Cummings, C., Brown, A., Ellison, J., Ohno, K., Rosenfeld, M. A., Tagle, D. A.
et al. (1997). Murine model of Niemann-Pick C disease:
mutation in a cholesterol homeostasis gene. Science
277,232
-235.
Malathi, K., Higaki, K., Tinkelenberg, A. H., Balderes, D. A.,
Almanzar-Paramio, D., Wilcox, L. J., Erdeniz, N., Redican, F., Padamsee, M.,
Liu, Y. et al. (2004). Mutagenesis of the putative
sterol-sensing domain of yeast Niemann Pick C-related protein reveals a
primordial role in subcellular sphingolipid distribution. J. Cell
Biol. 164,547
-556.
Min, K. T. and Benzer, S. (1999). Preventing
neurodegeneration in the Drosophila mutant bubblegum.
Science 284,1985
-1988.
Naureckiene, S., Sleat, D. E., Lackland, H., Fensom, A., Vanier,
M. T., Wattiaux, R., Jadot, M. and Lobel, P. (2000).
Identification of HE1 as the second gene of Niemann-Pick C disease.
Science 290,2298
-2301.
Neubueser, D., Warren, J. T., Gilbert, L. I. and Cohen, S.
M. (2005). molting defective is required for ecdysone
biosynthesis. Dev. Biol.
280,362
-372.[CrossRef][Medline]
Ono, H., Rewitz, K. F., Shinoda, T., Itoyama, K., Petryk, A.,
Rybczynski, R., Jarcho, M., Warren, J. T., Marques, G., Shimell, M. J. et
al. (2006). Spook and Spookier code for stage-specific
components of the ecdysone biosynthetic pathway in Diptera. Dev.
Biol. 298,555
-570.[CrossRef][Medline]
Palladino, M. J., Hadley, T. J. and Ganetzky, B.
(2002). Temperature-sensitive paralytic mutants are enriched for
those causing neurodegeneration in Drosophila.
Genetics 161,1197
-1208.
Parvy, J. P., Blais, C., Bernard, F., Warren, J. T., Petryk, A.,
Gilbert, L. I., O'Connor, M. B. and Dauphin-Villemant, C.
(2005). A role for betaFTZ-F1 in regulating ecdysteroid titers
during post-embryonic development in Drosophila melanogaster. Dev.
Biol. 282,84
-94.[CrossRef][Medline]
Patterson, M. C. (2003). A riddle wrapped in a
mystery: understanding Niemann-Pick disease, type C.
Neurologist 9,301
-310.[CrossRef][Medline]
Patterson, M. C. and Platt, F. (2004). Therapy
of Niemann-Pick disease, type C. Biochim. Biophys.
Acta 1685,77
-82.[Medline]
Petryk, A., Warren, J. T., Marques, G., Jarcho, M. P., Gilbert,
L. I., Kahler, J., Parvy, J. P., Li, Y., Dauphin-Villemant, C. and O'Connor,
M. B. (2003). Shade is the Drosophila P450 enzyme that
mediates the hydroxylation of ecdysone to the steroid insect molting hormone
20-hydroxyecdysone. Proc. Natl. Acad. Sci. USA
100,13773
-13778.
Robertson, H. M., Preston, C. R., Phillis, R. W.,
Johnson-Schlitz, D. M., Benz, W. K. and Engels, W. R. (1988).
A stable genomic source of P element transposase in Drosophila melanogaster.
Genetics 118,461
-470.
Somers, K. L., Brown, D. E., Fulton, R., Schultheiss, P. C.,
Hamar, D., Smith, M. O., Allison, R., Connally, H. E., Just, C., Mitchell, T.
W. et al. (2001). Effects of dietary cholesterol restriction
in a feline model of Niemann-Pick type C disease. J. Inherit.
Metab. Dis. 24,427
-436.[CrossRef][Medline]
Stocco, D. M. (2001). StAR protein and the
regulation of steroid hormone biosynthesis. Annu. Rev.
Physiol. 63,193
-213.[CrossRef][Medline]
Thummel, C. S. (1996). Files on steroids -
Drosophila metamorphosis and the mechanisms of steroid hormone action.
Trends Genet. 12,306
-310.[CrossRef][Medline]
Tsutsui, K., Ukena, K., Takase, M., Kohchi, C. and Lea, R.
W. (1999). Neurosteroid biosynthesis in vertebrate brains.
Comp. Biochem. Physiol.
124C,121
-129.
Warren, J. T., Sakurai, S., Rountree, D. B., Gilbert, L. I.,
Lee, S. S. and Nakanishi, K. (1988). Regulation of the
ecdysteroid titer of Manduca sexta: reappraisal of the role of the prothoracic
glands. Proc. Natl. Acad. Sci. USA
85,958
-962.
Warren, J. T., Petryk, A., Marques, G., Jarcho, M., Parvy, J.
P., Dauphin-Villemant, C., O'Connor, M. B. and Gilbert, L. I.
(2002). Molecular and biochemical characterization of two P450
enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA
99,11043
-11048.
Wismar, J., Habtemichael, N., Warren, J. T., Dai, J. D.,
Gilbert, L. I. and Gateff, E. (2000). The mutation without
children(rgl) causes ecdysteroid deficiency in third-instar larvae of
Drosophila melanogaster. Dev. Biol.
226, 1-17.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Starostina, A. Xu, H. Lin, and C. W. Pikielny A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein J. Biol. Chem., January 2, 2009; 284(1): 585 - 594. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Phillips, E. A. Woodruff III, P. Liang, M. Patten, and K. Broadie Neuronal Loss of Drosophila NPC1a Causes Cholesterol Aggregation and Age-Progressive Neurodegeneration J. Neurosci., June 25, 2008; 28(26): 6569 - 6582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Talamillo, J. Sanchez, R. Cantera, C. Perez, D. Martin, E. Caminero, and R. Barrio Smt3 is required for Drosophila melanogaster metamorphosis Development, May 1, 2008; 135(9): 1659 - 1668. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
