1 Departments of Developmental Biology, Genetics, and Bioengineering, Howard
Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA
94305-5439, USA
2 Department of Molecular and Cellular Physiology, Stanford University School of
Medicine, Stanford, CA 94305-5439, USA
* Author for correspondence (e-mail: scott{at}cmgm.stanford.edu)
Accepted 8 September 2005
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SUMMARY |
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Key words: Niemann-Pick Type C, Sterol, Steroid, Ecdysone, Drosophila, Lysosome storage
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Introduction |
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Neurons in individuals with NPC gene mutations and in a cat model of the
disease grow extra dendritic processes and form neurofibrillary tangles (NFTs)
(Walkley and Suzuki, 2004),
and progressive neurodegeneration is a prominent symptom, particularly in the
cerebellum (Higashi et al.,
1993
). Options for therapy are highly limited at this time
(Patterson and Platt, 2004
).
The outcome for individuals with NPC is usually death in the teenage years.
Understanding the origins of NPC is important to find ways to save lives and
because it provides an entry point for studying still-mysterious aspects of
lipid and transport cell biology.
The two NPC genes encode entirely different types of proteins that probably
participate in the same pathway, though the molecular mechanisms that link the
two proteins are unknown (Vanier and
Millat, 2003). NPC1, a cholesterol binding
(Ohgami et al., 2004
) 13
transmembrane region protein (Davies and
Ioannou, 2000
) is required for normal movements of populations of
late endosomes and for proper homeostatic regulation of sterol and other lipid
levels. Further interest in the NPC1 protein derives from its striking
sequence similarity to the Patched protein
(Carstea et al., 1997
), the
receptor for Hedgehog signaling proteins that regulate many aspects of growth
and cell fate determination during development
(Hooper and Scott, 2005
).
Another closely related protein, NPC1L1, is crucial for intestinal uptake of
cholesterol (Altmann et al.,
2004
; Davies et al.,
2005
). NPC1 works in a mysterious partnership with NPC2, a
lysosomal protein that can be secreted and that binds strongly to cholesterol
(Naureckiene et al., 2000
;
Friedland et al., 2003
;
Ko et al., 2003
). The central
mysteries still remain: what are the molecular functions of NPC1 and 2, how
does either one regulate organelle movements and molecular trafficking, and
why does loss of either protein lead to neurodegeneration and other
symptoms?
The npc1 gene is well conserved through about a billion years of
evolution, allowing studies with a variety of powerful experimental organisms
(Higaki et al., 2004). Useful
NPC1 models have been generated using yeast
(Malathi et al., 2004
) and
worm (Sym et al., 2000
),
although no sterol trafficking defect has been reported in either model. By
mutating one of the two NPC1-like fly genes, dnpc1a
(NPC1 FlyBase) we have generated a Drosophila model
of NPC1 that has a cholesterol accumulation defect similar to that of
mammalian NPC mutants. dnpc1a mutants accumulate sterol in a punctate
pattern in many tissues, implying a conserved role of NPC1 in cholesterol
trafficking from fly to mammals.
We find that dnpc1a function is crucial for normal steroid hormone
metabolism. Neurosteroid treatment has been shown to suppress
neurodegeneration in Npc1 mutant mice
(Griffin et al., 2004),
implying a neurosteroid hormone deficiency in Npc1 mice that may
parallel the defect in Drosophila. Our studies with dnpc1a
mutants suggest a model for NPC1 function: the protein may allow delivery of
sufficient sterol to mitochondria in order for steroid hormones to be made
there. More complete understanding of the molecular and cell biology of these
pathways may increase the options for NPC therapy.
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Materials and methods |
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Molecular biology
Full-length dnpc1a cDNA was amplified by RT-PCR and subcloned into
pBluescript SK. The stop codon was replaced with a KpnI site using
PCR and the new full-length cDNA was inserted in frame into the KpnI
site of pEYFP-N3 to create a dNPC1a-YFP fusion construct
(pEYFP-dnpc1a). From this clone, a 4.3 kb RI-NotI
fragment containing full-length dnpc1a cDNA and EYFP-coding region
was inserted into the pUAST vector to create the
pUAST-dnpc1a-yfp construct. All constructs requiring PCR
amplification were confirmed by sequencing.
RNA hybridization
dnpc1a and dnpc1b transcripts were detected by in situ
hybridization of full-length antisense and sense (control) probes to 0- to
16-hour-old embryos and wandering third instar larvae; detection was with
anti-DIG alkaline phosphatase and the CBIP/NBT substrate (Roche).
RNAi
dsRNA was prepared from PCR-generated template that corresponds to
nucleotides 3086-3603 of dnpc1a cDNA. dsRNA was synthesized with the
Ambion Megascript kit. Zero to 1 hour dechorionated embryos were injected
under oil with dsRNA at 2 µM.
Lethal phase determination
Each dnpc1a allele was balanced with CyO, P[w+, act-GFP].
The flies were allowed to lay eggs on apple juice plates, supplemented with
baker's yeast paste, at 25°C. Eggs were collected in 2-hour periods, and
embryos or larvae were identified as dnpc1a homozygotes by the
absence of the GFP-marked balancer chromosome. Larval stages were determined
by the appearance of the mouth hooks.
Sterol and ecdysone feeding
For dnpc1a mutants, each group of 200 first instar larvae was
placed on apple juice plates with baker's yeast paste containing supplement
sterols, and the lethal phases were determined. The final concentrations for
the sterols used were: cholesterol, 0.14 mg/g and 1.4 mg/g;
7-dehydrocholesterol, 0.014 mg/g, 0.14 mg/g and 1.4 mg/g; 20-hydroxyecdysone
(20E), 8 µg/g; ergosterol, 0.14 mg/g and 1.4 mg/g; progesterone, 1.4 mg/g;
and desmosterol, 1.4 mg/g.
Filipin staining and brain histology
For filipin staining, tissues were fixed in 4% paraformaldehyde for 30
minutes, washed twice in PBS, and stained with 50 µg/ml filipin (Sigma)
solution for 30 minutes. Samples were washed twice with PBS before mounted in
Vectashield mounting medium. All pictures were taken with a Zeiss compound
microscope with a DAPI filter.
The heads of mutant and wild-type flies, obtained 16-24 hours after eclosion, were removed from their bodies. The proboscis and ventral air sac were removed to aid fixation. The heads were placed in Carnoys fixative for 6 hours at room temperature. They were washed in 100% ethanol and embedded in HistoGel (Richard-Allan Scientific). The pellets containing the heads were processed through alcohol, xylene and molten Paraplast. They were embedded in Paraplast and 6 µm sections were cut. The tissue was stained with Haematoxylin and Eosin, and the slides mounted in Permount. Similar methods were used in making brains sections from first-instar larvae.
Electron microscopy
Dissected tissues from wild type and mutants were prepared for TEM using a
microwave protocol. We used a Pelco Laboratory microwave (model #3451)
equipped with a Cold Spot, Steadytemp chiller/recirculator run at 15°C and
vacuum chamber (Ted Pella, Mountain Lakes, CA). Malpighian tubules were fixed
in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4
in the microwave. Following two brief rinses in 0.1 M cacodylate buffer
containing 0.3 M sucrose on the bench, the tissue was post-fixed in the
microwave using 2% osmium tetroxide in 0.1 M cacodylate buffer containing 0.8%
potassium ferricyanide. After rinsing with distilled water, the tissue was
dehydrated in an ascending alcohol series in the microwave. The tissue was
infiltrated and embedded in Embed 812 resin (Electron Microscopy Sciences,
Fort Washington, PA). The tubule samples were oriented in a flat embedding
mold and hardened overnight in a 65°C oven. Semi-thin sections (1 to 2
µm) were cut using a Diatome HistoKnife (Diatome, USA) and stained with
Toludine Blue. Thin sections (50-70 nm) were cut with a Diatome diamond knife
and mounted on Formvar coated grids. The samples were examined on a JEOL 1230
electron microscope at 80 kV and photographed using a Gatan 967 slow-scan
cooled CCD camera.
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Results |
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In wandering third-instar larvae the dnpc1a RNA level is highest in the prothoracic gland component of the ring gland (Fig. 2F), and is accompanied by ubiquitous expression in other tissues including brain, garland cells, midgut and imaginal discs (Fig. 2F,G). 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.
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 (data not shown).
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Next, we produced loss-of-function dnpc1a mutations. 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 (Fig. 1B). The KG05670 transposon is closer to the 5' UTR of dnpc1a than to pros35 (Fig. 1B). Using KG05670 as a starting strain for imprecise excision, we generated two deletion alleles of dnpc1a that cause N-terminal 182 and 45 amino acid deletions. The alleles will be referred to as dnpc1a1 and dnpc1a2 (Fig. 1B). 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 (Fig. 2H).
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 (data not shown).
A dNPC1a function in sterol metabolism
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
(Friend and Bearer, 1981),
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 (Clark and Block,
1959
). 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.
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The first instar arrest of dnpc1a mutants can be prevented by ecdysone in the food
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
(Fig. 3A,B). 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)
(Gilbert et al., 2002
). Flies
reared on the ergosterol biosynthesis-defective yeast mutant, erg-6,
die during a prolonged first instar, similar to that of dnpc1a
(Parkin and Burnet, 1986
).
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, we performed 20-hydroxyecdysone-feeding experiments. 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.
Without 20E, 100% of dnpc1a homozygotes died 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) prevented 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; not shown), 45% died in the second instar and 2% died in the third instar (Fig. 3C). If the feeding with 20E was initiated late in the first instar (40 hours AEL), the rescue was similar but weaker (Fig. 3C). The results indicate that the first instar arrest of dnpc1a mutants is likely to be a consequence of insufficient ecdysone.
High cholesterol or 7-dehydrocholesterol in the medium rescues dnpc1a mutants
For insect ecdysone biosynthesis, the substrate cholesterol is first
converted to 7-dehydrocholesterol, probably by a microsomal/endoplasmic
reticulum (ER)-localized P450 enzyme
(Gilbert et al., 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
(Gilbert et al., 2002
).
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, we increased the sterol concentration in the yeast paste.
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)
(Xu and Nes, 1988
). 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 (Fig. 4A).
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.
Ergosterol is able to support the growth and reproduction of
Drosophila (Clark and Block,
1959). 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 (Fig. 4A).
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.
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
(Fig. 4B). A significant
percentage of rescued flies even reach adulthood, although they usually died
within a day or two after eclosion (Fig.
4B). 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 et al., 2002), or
with progesterone, a human steroid hormone derived from cholesterol, did not
rescue at all (Fig. 4B).
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No obvious neurodegeneration in dnpc1a mutants
As the Drosophila dnpc1a model recapitulates the sterol
accumulation phenotypes of NPC disease, we investigated whether the flies also
have a neurodegeneration problem, another characteristic of mammalian NPC
disease. Drosophila neurodegeneration mutants often have a short life
span and numerous large vacuoles in brain
(Min and Benzer, 1999;
Palladino et al., 2002
;
Tschape et al., 2003
).
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. We sectioned brains from
7-dehydrocholesterol-treated dnpc1a sick adult escapers before they
died. The gross brain morphology is fine in the mutants and there were no
evidence of neurodegenerative vacuoles in the brain sections
(Fig. 5). 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 (data not shown). We cannot exclude the possibility
that neurodegeneration may happen in small subset(s) of neurons.
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We investigated 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.
We used the UAS-Gal4 system 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 (Table 1). 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 (Table 1).
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dnpc1a larval and adult phenotypes in flies with ring gland-specific expression of dNPC1a-YFP
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
(Fig. 6). We directly compared
sterol accumulation 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
(Fig. 6C,E). dnpc1a
mutants rescued by ecdysone feeding have sterol accumulation in both ring
glands and brains (Fig. 6D,F),
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 (Fig.
6G,H).
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 (Fig.
7A,B). 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
(Fig. 7C,D). 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
NPC1 mutations (Pellissier et
al., 1976). Excess sterol that accumulates because of lipid
trafficking defects may be stored in similar aberrant organelles in
Drosophila and mammals. Our sterol supplementation data suggest that
the sterol in those multi-lamellar organelles is not available for
synthesizing steroid hormones.
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Discussion |
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Relations of NPC1 function to steroid synthesis
Given the evident steroid hormone synthesis defect in dnpc1a flies
and the neurosteroid deficiency in Npc1/Npc1 mice, the role of NPC1
in steroid hormone biosynthesis becomes a central mystery. The steroid
biosynthesis pathways are generally very similar in vertebrates and insects.
In mammals, the substrate, cholesterol, is delivered to the outer
mitochondrial membrane and then to the inner membrane. In the insect steroid
synthesis pathway, dietary cholesterol is first converted to
7-dehydrocholesterol in the ER, then translocated to the mitochondria by an
unknown mechanism (Gilbert et al.,
2002). Once in the inner mitochondrial compartment, the sterol
(cholesterol in vertebrates, 7-dehydrocholesterol in insects) is converted
into different steroid hormones through a series of enzymatic reactions
carried out by P450 enzymes. NPC1 protein has not been observed in
mitochondria (Higgins et al.,
1999
; Neufeld et al.,
1999
; Ko et al.,
2001
), so it may function in a delivery process. Together, our
studies and others point in one direction: the need for NPC1 to ensure that
sufficient intracellular cholesterol substrate is available for steroid
hormone biosynthesis. In NPC mutants, cholesterol accumulates in aberrant
endosome or lysosome-like compartments. We hypothesize that this trapping
process may cause or reflect a deficiency of cholesterol in other
compartments, such as ER and/or mitochondria, resulting in deficient steroid
hormone synthesis.
A recent study showed that mitochondria from whole brain preparations of
Npc1 mutant mice contain more cholesterol than similar extracts from
wild-type animals (Yu et al.,
2005). This apparent contradiction to the cholesterol deficiency
model could be due to pooling different cell types, such as diverse neurons
that do not synthesize cholesterol, with cells that do, thus masking a sterol
deficiency in the mitochondria of critical cell types. Alternatively, standard
mitochondria preparations usually contain some endosomes and lysosomes. The
cholesterol richness of endosomes and lysosomes might have masked a
cholesterol deficit in mitochondria. The masking may be particularly severe in
npc1 mutants as there is massive accumulation of cholesterol in
mutant endosomes and lysosomes.
Mammals synthesize numerous steroid hormones that have a wide range of
important physiological functions. No defect in general steroidogenesis has
been observed in NPC1-deficient humans or mice
(Soccio and Breslow, 2004);
only neurosteroids are lacking from Npc1 mutant mice
(Griffin et al., 2004
). Why is
neurosteroid biosynthesis particularly sensitive to the loss of NPC1 function?
The answer may come from the source of cholesterol for mitochondria and the
route for moving cholesterol to mitochondria. In mammals, the source of
cholesterol for mitochondrial steroidogenesis varies between cell type or
condition: some cells use HDL cholesterol esters in lipid droplets, others use
LDL cholesterol arriving via the endosomal pathway, and still others obtain
cholesterol by de novo synthesis from acetate
(Soccio and Breslow, 2004
).
The differing sources and pathways may make NPC1 more important for steroid
synthesis in some cell types than in others.
Critical requirement for NPC1 in different tissues in different organisms
Purkinje neurons, which undergo prominent neurodegeneration in NPC disease,
are the main cells for neurosteroid biosynthesis in the brain
(Tsutsui et al., 1999). Using
chimeric mice in which some cells have Npc1 function and others do
not, we have shown that Npc1 is required within Purkinje neurons for
their survival (Ko et al.,
2005
). The apparent cell autonomous role of NPC1 in Purkinje cells
raises a question: if NPC1 is required only for neurosteroid biosynthesis, why
do wild-type cells fail to help their mutant neighbors? One explanation could
be an unidentified function of NPC1 in Purkinje cells in addition to
controlling neurosteroid biosynthesis. Alternatively, there may be a
cell-autonomous autocrine neurosteroid signal in Purkinje cells.
Like mammalian NPC1, Drosophila dnpc1a is widely expressed, but our results show that the crucial function of dnpc1a for development into an adult is restricted to a single tissue (the ring gland), and to a specific biological process (ecdysone biosynthesis).
NPC: a sterol `shortage' disease?
If the steroid synthesis hypothesis about NPC is correct, the disease
should be regarded more as a sterol shortage disease than a sterol excess
disease, because the accumulated sterol embedded in multi-lamellar membranes
is evidently unavailable for further sterol metabolism. The cholesterol
shortage model clearly differs from models in which the lipid accumulation
itself is the cause of disease pathology. In fact, individuals with NPC live
for many years carrying significant accumulations of lipids, including sterols
and gangliosides, in many cells. Adult Drosophila dnpc1a homozygotes,
rescued by ring gland dnpc1a expression, seem fully functional except
for male sterility, despite the punctate sterol accumulation in many of their
tissues. It remains unclear how much damage the accumulated sterol and other
lipids impart.
The sterol shortage model is also supported by studies of mammalian NPC1
disease, where the transcriptional program for sterol biosynthesis involving
the SREBP transcription factor is triggered in cells that are replete with
sterol (Liscum and Faust,
1987). Normally such sterol abundance would leave SREBP tethered
in the ER. SREBP is activated by a regulatory system that senses sterol level
in the ER and, when sterol seems low, allows SREBP to move into the Golgi and
then to the nucleus where it triggers transcription of genes for sterol
synthesis, such as HMG CoA reductase, and genes for sterol import, such as LDL
receptor (Brown and Goldstein,
1997
). The activation of SREBP in Npc1 mutant cells,
despite the abundant sterol, shows that the accumulated sterol is invisible to
the cells' regulatory machinery.
Our studies suggest possible new directions for improving NPC disease
therapy. Previous therapeutic efforts in lowering cholesterol level and
limiting dietary cholesterol supply have been unsuccessful
(Akaboshi and Ohno, 1995;
Somers et al., 2001
), as would
be expected if the accumulation of sterol in cells is not the primary cause of
pathology. Instead, our results point in the opposite direction: the disease
might be usefully treated by increasing the cholesterol level in the
mitochondria of critical types of neurons.
The evolutionary conservation of sterol accumulation in Npc1 mutants of flies and mammals implies a common trafficking function that existed at least half a billion years ago. The further possible similarity in disease mechanisms that is implied by the steroid deficit in Drosophila and mice NPC model may permit advances to be made in understanding mammalian disease by employing classical genetic approaches in flies.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Akaboshi, S. and Ohno, K. (1995). Niemann-Pick disease type C. Nippon Rinsho. 53,3036 -3040.[Medline]
Altmann, S. W., Davis, H. R., Jr, Zhu, L. J., Yao, X., Hoos, L.
M., Tetzloff, G., Iyer, S. P., Maguire, M., Golovko, A., Zeng, M. et
al. (2004). Niemann-Pick C1 Like 1 protein is critical for
intestinal cholesterol absorption. Science
303,1201
-1204.
Brown, M. S. and Goldstein, J. L. (1997). The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89,331 -340.[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.
Clark, A. J. and Block, K. (1959). The absence
of sterol synthesis in insects. J. Biol. Chem.
234,2578
-2582.
Davies, J. P. and Ioannou, Y. A. (2000).
Topological analysis of Niemann-Pick C1 protein reveals that the membrane
orientation of the putative sterol-sensing domain is identical to those of
3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding
protein cleavage-activating protein. J. Biol. Chem.
275,24367
-24374.
Davies, J. P., Scott, C., Oishi, K., Liapis, A. and Ioannou, Y.
A. (2005). Inactivation of NPC1L1 causes multiple lipid
transport defects and protects against diet-induced hypercholesterolemia.
J. Biol. Chem. 280,12710
-12720.
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.
Friend, D. S. and Bearer, E. L. (1981). beta-Hydroxysterol distribution as determined by freeze-fracture cytochemistry. Histochem. J. 13,535 -546.[CrossRef][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]
Higgins, M. E., Davies, J. P., Chen, F. W. and Ioannou, Y. A. (1999). Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol. Genet. Metab. 68, 1-13.[CrossRef][Medline]
Hooper, J. E. and Scott, M. P. (2005). Communicating with Hedgehogs. Nat. Rev. Mol. Cell. Biol. 6,306 -317.[CrossRef][Medline]
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. 1,81 -95.[Medline]
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.
Liscum, L. and Sturley, S. L. (2004). Intracellular trafficking of Niemann-Pick C proteins 1 and 2, obligate components of subcellular lipid transport. Biochim. Biophys. Acta 1685,22 -27.[Medline]
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.
Matyash, V., Entchev, E. V., Mende, F., Wilsch-Brauninger, M., Thiele, C., Schmidt, A. W., Knolker, H. J., Ward, S. and Kurzchalia, T. V. (2004). Sterol-derived hormone(s) controls entry into diapause in Caenorhabditis elegans by consecutive activation of DAF-12 and DAF-16. PLoS Biol. 2,1561 -1571.
Min, K. T. and Benzer, S. (1999). Preventing
neurodegeneration in the Drosophila mutant bubblegum.Science 284,1985
-1988.
Mukherjee, S. and Maxfield, F. R. (2004). Lipid and cholesterol trafficking in NPC. Biochim. Biophys. Acta 1685,28 -37.[Medline]
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.
Neufeld, E. B., Wastney, M., Patel, S., Suresh, S., Cooney, A.
M., Dwyer, N. K., Roff, C. F., Ohno, K., Morris, J. A., Carstea, E. D.
et al. (1999). The Niemann-Pick C1 protein resides in a
vesicular compartment linked to retrograde transport of multiple lysosomal
cargo. J. Biol. Chem.
274,9627
-9635.
Ohgami, N., Ko, D. C., Thomas, M., Scott, M. P., Chang, C. C.
and Chang, T. Y. (2004). Binding between the
Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a
functional sterol-sensing domain. Proc. Natl. Acad. Sci.
USA 101,12473
-12478.
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.
Parkin, C. A. and Burnet, B. (1986). Growth arrest of Drosophila melanogaster on erg-2 and erg-6 sterol mutant strains of Saccharomyces cerevisiae. J. Insect Physiol. 32,463 -471.[CrossRef]
Patterson, M. C. and Platt, F. (2004). Therapy of Niemann-Pick disease, type C. Biochim. Biophys. Acta 1685,77 -82.[Medline]
Pellissier, J. F., Hassoun, J., Gambarelli, D., Bryon, P. A., Casanova, P. and Toga, M. (1976). [Niemann-Pick disease (Crocker's type C): ultrastructural study of a case (author's transl)]. Acta Neuropathol. 34, 65-76.[CrossRef][Medline]
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.
Soccio, R. E. and Breslow, J. L. (2004).
Intracellular cholesterol transport. Arterioscler. Thromb. Vasc.
Biol. 24,1150
-1160.
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]
Sturley, S. L., Patterson, M. C., Balch, W. and Liscum, L. (2004). The pathophysiology and mechanisms of NP-C disease. Biochim. Biophys. Acta 1685,83 -87.[Medline]
Sym, M., Basson, M. and Johnson, C. (2000). A model for niemann-pick type C disease in the nematode Caenorhabditis elegans. Curr. Biol. 10,527 -530.[CrossRef][Medline]
Timmons, L., Becker, J., Barthmaier, P., Fyrberg, C., Shearn, A. and Fyrberg, E. (1997). Green fluorescent protein/beta-galactosidase double reporters for visualizing Drosophila gene expression patterns. Dev. Genet. 20,338 -347.[CrossRef][Medline]
Tschape, J. A., Bettencourt da Cruz, A. and Kretzschmar, D. (2003). Progressive neurodegeneration in Drosophila: a model system. J. Neural. Transm. 65, 51-62.[CrossRef]
Tsutsui, K., Ukena, K., Takase, M., Kohchi, C. and Lea, R. W. (1999). Neurosteroid biosynthesis in vertebrate brains. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 124,121 -129.[CrossRef][Medline]
Vanier, M. T. and Millat, G. (2003). Niemann-Pick disease type C. Clin. Genet. 64,269 -281.[CrossRef][Medline]
Walkley, S. U. and Suzuki, K. (2004). Consequences of NPC1 and NPC2 loss of function in mammalian neurons. Biochim. Biophys. Acta 1685,48 -62.[Medline]
Xu, S. H. and Nes, W. D. (1988). Biosynthesis of cholesterol in the yeast mutant erg6. Biochem. Biophys. Res. Commun. 155,509 -517.[CrossRef][Medline]
Yu, W., Gong, J. S., Ko, M., Garver, W. S., Yanagisawa, K. and
Michikawa, M. (2005). Altered cholesterol metabolism
in Niemann-Pick type C1 mouse brains affects mitochondrial function.
J. Biol. Chem. 280,11731
-11739.
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