Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
Author for correspondence (e-mail:
jht{at}u.washington.edu)
Accepted 18 August 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dauer, Niemann-Pick type C disease, Cholesterol, Hormone, C. elegans
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The C. elegans genome contains two homologs of the human NPC1
gene, ncr-1 and ncr-2 (NPC1 related genes), formerly known
as npc-1 and npc-2, respectively
(Sym et al., 2000). Both
ncr gene products have 31% identity with human NPC1 and contain all
the structural domains of NPC1 (Sym et
al., 2000
), including an N-terminal NPC1 domain and a
sterol-sensing domain (SSD) (Chun and
Simoni, 1992
). C. elegans requires cholesterol in the
growth medium (Chitwood,
1999
), but little is known about the biological pathways mediating
the uptake of cholesterol and subsequent processing and distributing steps
(Kurzchalia and Ward, 2003
).
Nonetheless, the sequence conservation between human NPC1 and worm
ncr genes suggests that an intracellular sterol trafficking pathway
might be conserved.
A C. elegans model for NP-C1 disease was established by creating
deletion mutations in both Ncr genes. Both deletions cause early frameshift
mutations, and are thus likely to be null alleles. Although the single
ncr-1 and ncr-2 mutants have subtle phenotypes, the
ncr-2; ncr-1 double mutant has a strong dauer formation constitutive
(Daf-c) phenotype (Sym et al.,
2000).
C. elegans dauers are alternative third-stage larvae that form in
response to stressful environmental cues
(Cassada and Russell, 1975;
Riddle and Albert, 1997
).
Mutations in dauer formation (daf) genes result in either failure to
form dauers in response to dauer inducing stimuli (dauer formation defective,
or Daf-d), or constitutive formation of dauer larvae (dauer formation
constitutive, or Daf-c). The daf genes function in complex parallel
signaling pathways, including a cGMP pathway
(Ailion and Thomas, 2000
;
Birnby et al., 2000
;
Coburn and Bargmann, 1996
;
Komatsu et al., 1996
), a
TGF-ß signaling pathway (Ren et al.,
1996
; Schackwitz et al.,
1996
) and an insulin pathway
(Kimura et al., 1997
;
Li et al., 2003
;
Lin et al., 1997
;
Ogg et al., 1997
). The outputs
of these pathways are thought to be integrated by the nuclear hormone receptor
DAF-12. Recently, elements of a hormonal pathway that probably acts on DAF-12
have been identified. They include a cytochrome P450 enzyme encoded by
daf-9 (Gerisch et al.,
2001
; Jia et al.,
2002
) and a PTP (protein tyrosine phosphatase)-like protein
encoded by sdf-9 (Ohkura et al.,
2003
). The DAF-9 protein is hypothesized to catalyze a step in the
biosynthesis of a sterol ligand that inhibits the dauer promoting activity of
the DAF-12 receptor. Loss-of-function mutants of daf-9 share the
pleiotropic developmental phenotypes of class 6 alleles of daf-12,
which are thought to abolish ligand binding
(Antebi et al., 1998
;
Antebi et al., 2000
). SDF-9 is
thought to augment or facilitate DAF-9 function
(Ohkura et al., 2003
).
daf-9 and sdf-9 are co-expressed in a pair of head cells
with possible neuroendocrine characteristics, called XXX cells
(Ohkura et al., 2003
). Killing
the XXX cells induced dauer formation in wild-type animals and this cellular
function was mapped to the same position in the dauer pathway as mutations in
sdf-9 (Gerisch et al.,
2001
; Ohkura et al.,
2003
).
We present evidence that ncr-1 and ncr-2 are involved in
processing cholesterol, and that they function upstream of daf-9 and
daf-12 in regulating dauer formation. We demonstrate that the
expression pattern of ncr-1 largely coincides with the pattern of
cholesterol tissue distribution in C. elegans
(Matyash et al., 2001;
Merris et al., 2003
), while
the expression of ncr-2 is more restricted. Both ncr-1 and
ncr-2 are expressed in the XXX cells. The ncr-1 mutant is
sensitive to cholesterol deprivation (Sym
et al., 2000
) and to progesterone, an inhibitor of intracellular
cholesterol trafficking. We propose that ncr-1 is involved in sterol
trafficking in general and that ncr-1 and ncr-2 function
together in the XXX cells to affect the synthesis of the dauer-regulating
sterol hormone.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assays
When continuously propagated, the ncr-2; ncr-1 strain became
progressively less healthy. This effect was apparent in poor recovery from the
dauer and adult sterility. By contrast, worms that were freshly recovered from
starved plates were much healthier and fecund. A similar phenomenon was
reported for heterochronic mutants (Antebi
et al., 1998; Liu and Ambros, 1991). In order to minimize
variability between experiments, all assays involving the ncr-2;
ncr-1 strain used worms that were recovered from starved plates or their
progeny. For example, in media sterol assays, ncr-2; ncr-1 mutants
from starved NGM plates were allowed to recover on the assay plates, and noted
as the starting generation (Gen 0). Dauer formation was scored for Gen 1 and
Gen 2 progeny. Gen 1 progeny were also scored for dauer recovery, life span
and brood size. Control strains in the same assays were treated in the same
way.
For dauer formation assays, 6-10 adult hermaphrodites were allowed to lay
eggs at room temperature (about 22°C) for 2-6 hours and then incubated at
the assay temperature. Dauer and non-dauer animals were scored after 72-80
hours at 20°C, 54-60 hours at 25°C and 44-48 hours at 27°C. Brood
size and life span assays were conducted at 20°C as described
(Gems et al., 1998), for all
strains except ncr-2; ncr-1. The zero time point was the time of the
L4 transfer. The number of progeny was scored daily and then summed to get the
total brood size. Similar procedures were followed for ncr-2; ncr-1,
except that dauers from staged egg-lays were used in the assays instead of L4
larvae. In this case, the zero point was the time of dauer transfer. Samples
that died from internal hatching of embryos or from exploded vulva were
included in the graphs. Statistical analyses including Student's
t-test and the Kruskal-Wallis nonparametric ANOVA test were performed
using InStat2.01.
Non-complementation screen for new ncr-1 alleles
ncr-2(nr2023) males were mutagenized by EMS
(Sulston and Hodgkin, 1988),
and allowed to recover overnight. These males were crossed to
ncr-2(nr2023); ncr-1(nr2022) lon-2(e678) or to ncr-2(nr2023);
ncr-1(nr2022) lon-2(e678) daf-12(m20). Non-Lon dauers were picked in the
F1 and new ncr-1 alleles were made homozygous by selecting F2 animals
that segregated no Lon progeny.
Sequencing and cloning cDNAs
The gene structure of ncr-1 was deduced by sequencing the yk39e8
cDNA clone. The gene structure of ncr-2 was deduced by sequencing
RT-PCR products generated by gene specific primers. The 5' end of both
genes were determined by sequencing PCR products amplified with SL1 leader
sequence and gene specific primers. For ncr-2, the 3' end was
determined by sequencing 3' RACE products amplified by gene specific
primers and adapter primers (Invitrogen).
Germline transformation
Germline transformation was carried out as described
(Mello et al., 1991), at a
concentration of 5-50 ng/ml test DNA with 5-50 ng/ml co-injection marker
[myo-2p::gfp, or pBLH98(lin-15+)] and 50-80 ng/ml
carrier DNA [pBluescript KS(+)].
Single gene rescue and reporter constructs
A 13 kb KpnI fragment of cosmid F02E8 containing the
ncr-1A coding region with 3288 bp of upstream promoter sequence was
sufficient to rescue the Daf-c phenotype of the ncr-2; ncr-1 mutant
(see Table S1 in the supplementary material). A PCR fragment containing the
ncr-2 coding region, with 1084 bp of upstream promoter sequence, was
sufficient to rescue the Daf-c phenotype of the ncr-2; ncr-1 mutant
(see Table S1 in the supplementary material). A PCR fragment containing the
daf-9a coding region with 1947 bp of promoter sequence was used for
DAF-9 overexpression studies.
The vectors pPD95.75 and TJ1665 (the dsRed2 derivative of pPD95.69) were used to study the gene expression patterns. pTJ1665 was made by substituting dsRed2 cDNA for the GFP-coding sequence of pPD95.69, removing the nuclear localization signal of pPD95.69 in the process. A 3540 bp fragment of ncr-1A promoter was amplified and cloned into the SphI and XmaI sites of pPD95.75 to generate plasmid pTJ1663 [ncr-1Ap(s)::gfp]. A longer 8709 bp fragment of ncr-1A promoter was used similarly to generate plasmid pTJ1726 [ncr-1Ap(l)::gfp]. For the rarer ncr-1B transcript, a 6571 bp fragment of ncr-1B promoter was amplified and cloned into pPD95.75 to generate plasmid pTJ1727(ncr-1Bp::gfp). For ncr-2, a 4568 bp promoter fragment was amplified and cloned into pPD95.75 to make plasmid pTJ1664 (ncr-2p::gfp). For plasmid pTJ1728 (ncr-2p::dsRed2), the PstI-XmaI promoter fragment from pTJ1664 was blunted at the PstI site and cloned into the HincII and XmaI sites of pTJ1665.
Behavioral assays
Osmotic avoidance assays were performed as described
(Culotti and Russell, 1978) by
testing whether animals crossed a ring of 8 M glycerol during 10 minute
assays. Chemotaxis toward the volatile attractive odorants and NaCl was
assayed as described (Bargmann et al.,
1993
; Bargmann and Horvitz,
1991
).
Imaging
Fluorescent dye-filling assays were performed as described
(Fujiwara et al., 1999;
Hedgecock et al., 1985
) using
DiI-C12 (Molecular Probes, Eugene, OR). Dye-filled animals were observed at
1000x magnification by conventional fluorescence microscopy (Zeiss
Axioskop). To capture the 3D morphology of ASER neuron, freshly formed dauer
larvae of the ncr-2; ncr-1 genotype and wild-type L3 larvae were set
up for observation as described (Swoboda
et al., 2000
). 3D data stacks were acquired in the FITC channel by
moving the focal plane in 0.5 µm increments through the entire worm.
Background and out-of-focus signal were removed using a conservative,
reiterative (15x) deconvolution algorithm
(Agard et al., 1989
;
Scalettar et al., 1996
). 3D
data stacks were combined using maximum intensity projections of all data
points along the z-axis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
High media cholesterol rescues ncr-2; ncr-1 mutants
Because the NPC proteins are involved in trafficking sterols in mammals, we
tested whether raising the media cholesterol concentration could rescue the
ncr-2; ncr-1 mutant. The Dafc phenotype of the ncr-2; ncr-1
mutant was significantly suppressed by raising the level of cholesterol in the
media (Fig. 2A). High
cholesterol also promoted recovery from dauer arrest and ameliorated the
post-dauer phenotypes, as demonstrated by improved brood size and lengthened
average life span (Fig. 2B,C).
Increasing media cholesterol had no obvious effect on the dauer formation
phenotype of other Dafc mutants, including daf-9, daf-2 and
daf-7 (Fig. 2A), though the recovery of daf-9 dauers was mildly promoted
(Jia et al., 2002) (data not
shown). These results suggest that cholesterol availability is limiting in
ncr-2; ncr-1, but not in the other Dafc mutants, consistent with an
NCR function in delivering sterol substrates to the DAF-9 enzyme.
In order to identify possible substrates and products of the DAF-9 enzyme,
we tested the ability of other sterols and steroids to suppress the Daf-c
phenotype of the ncr-2; ncr-1 mutant. Because it is abundant in fungi
and plants and can substitute for cholesterol to sustain the growth and
reproduction of the worm (Chitwood,
1999), ergosterol is a likely sterol source for C.
elegans in the wild. Pregnenolone is the product of the first side-chain
cleavage of cholesterol, the rate-limiting step in mammalian steroid
biosynthesis (Simpson, 1979
).
We also tested the insect molting hormone ecdysone and a human steroid hormone
progesterone. Only ergosterol moderately rescued the Daf-c phenotype of
ncr-2; ncr-1, whereas ecdysone and pregnenolone had no effect (data
not shown). Intriguingly, adding progesterone to normal NGM plates retarded
the larval development of the ncr-2; ncr-1 mutant and inhibited its
recovery from the dauer stage (data not shown). We decided to further examine
the effect of progesterone.
ncr-1 mutants are sensitive to progesterone in the growth media
Progesterone can inhibit intracellular cholesterol trafficking in mammalian
cells (Butler et al., 1992;
Liscum and Munn, 1999
). In
fibroblast cell cultures, it causes accumulation of unesterified cholesterol
in abnormal lysosome-like compartments, the same cellular phenotype that
characterizes NP-C1 disease. Multiple steps of cholesterol intracellular
trafficking appear to be affected by progesterone, but the exact mechanism and
the targets of its action remain unknown.
To determine whether progesterone also disturbs cholesterol trafficking in
C. elegans, we examined its effect on wild-type worms. Wild-type
worms grown on NGM plates that contained 20 µg/ml progesterone in addition
to the normal 5 µg/ml cholesterol developed into fertile adults. However,
when progesterone was added to low-sterol NGM plates, pleiotropic phenotypes
similar to those caused by cholesterol deprivation
(Gerisch et al., 2001;
Merris et al., 2003
) were
observed (Fig. 3). Many animals
were defective in gonadal tip cell migration (54%, n=37), with both
gonadal arms extending ventrally, failing to make the programmed turns
(Fig. 3B). Frequently, sperm
were observed in the pseudocoelomic space (39%, n=23), probably owing
to a disrupted basement membrane (Fig.
3D). Occasional dauer larvae were also observed.
|
|
|
|
Expression patterns of ncr-1 and ncr-2 genes
We used promoter GFP fusions to study the expression patterns of the
ncr-1 and ncr-2 genes. Three reporter constructs,
ncr-1Ap(s)::gfp, ncr-1Ap(l)::gfp and ncr-1Bp::gfp were made
for ncr-1 because of its complex gene structure
(Fig. 5B). The results indicate
that ncr-1 expression is widespread
(Fig. 5B) and largely coincides
with the reported distribution pattern of cholesterol in C. elegans,
which includes the following tissues: intestine, pharynx, excretory gland
cell, nerve ring, spermatheca and germ cells, including both oocytes and sperm
(Matyash et al., 2001;
Merris et al., 2003
). In the
following sections, we summarize the observed ncr-1 expression
pattern within specific tissues.
Intestine
Both ncr-1Ap(s)::gfp and ncr-1Ap(l)::gfp were strongly
expressed throughout the intestine (Fig.
6A), with posterior intestinal expression consistently stronger
than anterior expression.
|
Nervous system
ncr-1Ap(s)::gfp and ncr-1Ap(l)::gfp were expressed in the
same set of head and tail neurons and a pair of neuron-like cells identified
as the XXX cells (Fig. 6B,H)
(Ohkura et al., 2003).
According to their location and cellular morphology, we identified the head
neurons as the pharyngeal neuron I6, the inner labial sensory neurons IL2s and
the amphid neurons ASE and ASG. The expression level in the amphid neurons was
weaker than in the other head neurons. The tail neurons were identified as
PHC, in which expression was first detected during the L2 stage, consistent
with the time of birth of the neurons at the end of L1. In contrast to the
widespread expression of ncr-1Ap::gfp, ncr-1Bp::gfp is expressed
exclusively in 10-12 pairs of head and tail neurons
(Fig. 6K). The tail neurons
were identified as PHA, PHB and DVC. One pair of head neurons was identified
as AWC. The other head neurons were very tentatively identified as RIC, RIM,
FLP, ADA, ADE, RID and maybe AIY. We also occasionally observed expression in
a pair of head cells anterior to the nerve ring. The position and morphology
of these cells are similar to the XXX cells.
With the exception of PHC neurons, expression in the tissues above was first observed during late embryogenesis and did not change during development.
Somatic gonad
Both ncr-1Ap(s)::gfp and ncr-1Ap(l)::gfp were strongly
expressed in the spermatheca and weakly in the gonadal sheath cells
(Fig. 6D). The expression in
the somatic gonad could be observed only in adults.
Epidermis
ncr-1Ap(l)::gfp was strongly expressed in the excretory cell
(Fig. 6A-C) and rectal
epithelial cells (Fig. 6C) from
the early L1 larval stage and through all life stages. Seam cell expression
was first observed in the late L1 stage
(Fig. 6E), while expression in
the lateral hypodermis increased during the L3 stage and peaked during the L4
stage (Fig. 6F). Seam cell and
hypodermal expression gradually decreased in the adult stage and was hardly
visible among older adults (Fig.
6G). ncr-1Ap(s)::gfp was not expressed in the hypodermis
under normal growth conditions, though lateral hypodermal but not seam cell
expression was dramatically upregulated in starved animals of all
developmental stages (Fig.
6I,J). Similar upregulation of hypodermal expression in response
to starvation has been reported for daf-9
(Gerisch and Antebi, 2004). No
increase in hypodermal expression was seen in starved ncr-1Ap(l)::gfp
animals.
In contrast to the widespread expression of ncr-1, the expression of ncr-2p::gfp was restricted to the XXX cells and the somatic gonad. It was strongly expressed in the proximal gonadal sheath cells and weakly in the spermatheca (Fig. 6L). The XXX expression of ncr-2p::gfp was seen throughout development (Fig. 6M), while the gonadal expression was observed only in adults.
Position of ncr genes in the dauer formation pathway
To determine the position of ncr genes in the dauer pathway, we
analyzed the dauer formation phenotypes of daf-d; ncr-2; ncr-1 triple
mutants (Table 2). Mutations in
osm-6, daf-5 and daf-16 suppress the Daf-c phenotype of
mutants in the cGMP, TGF-ß and insulin pathways, respectively
(Thomas et al., 1993;
Vowels and Thomas, 1992
). The
Daf-c phenotype of ncr-2; ncr-1 was not suppressed by osm-6
and daf-5 mutations, placing the ncr genes parallel to or
downstream of these genes. A severe loss-of-function allele of daf-16,
m27, did not suppress the ncr-2; ncr-1 Daf-c phenotype, but the
null allele mgDf50 did suppress partially, suggesting that
daf-16 might be a minor downstream target of the ncr genes.
The loss-of-function allele daf-12(m20), completely
suppressed the Daf-c and the pleiotropic post-dauer phenotypes of ncr-2;
ncr-1 mutant (Fig. 2B,C),
placing the function of the ncr genes upstream of daf-12 and
also suggesting that the pleiotropic post-dauer phenotypes of ncr-2;
ncr-1 result from unregulated DAF-12 activity. This pathway position for
the ncr genes is the same as daf-9
(Gerisch et al., 2001
;
Jia et al., 2002
), suggesting
that the ncr genes function in the hormonal branch of the dauer
formation pathway.
|
|
|
A morphological defect was observed in the ASER neuron
(gcy-5::gfp) (Yu et al.,
1997): during the transient dauer stage, ASER often (69.2%,
n=13) had an ectopic posterior process terminating in one or more
swellings (Fig. 7A). The defect
appeared to be transient because it was absent during the early larval stages,
peaked during dauer stage, then regressed as dauers recovered
(Fig. 7B). This structural
change was not observed in dauers formed by wild-type worms on starved plates,
nor in dauers formed by the daf-7(e1372) mutant, and therefore was
not caused by dauer arrest itself. Moreover, this morphological defect in ASER
was not observed in ncr-1 or ncr-2 single mutants growing
reproductively or in dauers induced by starvation, suggesting that it is
specifically associated with the transient dauer stage of the ncr-2;
ncr-1 double mutant.
|
ASE and the dye-filled head neurons are all amphid neurons. We examined
other types of neurons, including interneuron AIY (ttx-3::gfp)
(Hobert et al., 1997), sensory
neurons BAG (gcy-33::gfp) (Yu et
al., 1997
), IL2 (ncr-1Ap(s)::gfp) and GABAergic
motoneurons (unc-25::gfp) (Jin et
al., 1999
). A small proportion (18%, n=33) of ncr-2;
ncr-1 mutant animals exhibited an AIY defect similar to that observed in
ASER during the transient dauer stage. No defect was observed in the other
neuron classes during or after the transient dauer stage. We conclude that NCR
function is required to maintain the morphological integrity of some but not
all neurons during the transient dauer stage and that amphid neurons as a
class may be more severely affected than other neurons.
Many amphid neurons function in regulating dauer formation and in
chemotaxis response to soluble and volatile environmental stimuli, including a
major role for ASE neurons in chemotaxis towards NaCl
(Bargmann and Horvitz, 1991).
To assess the function of amphid neurons, we measured the avoidance response
of ncr-2; ncr-1 adults to high osmotic strength and their chemotaxis
response to NaCl, diacetyl, isoamyl alcohol, and 2, 4, 5-trimethylthiazole
(Bargmann et al., 1993
;
Bargmann and Horvitz, 1991
).
ncr-2; ncr-1 mutants exhibited robust responses towards all the above
stimuli, indicating normal neuronal function. However, the ncr-2;
ncr-1 adult animals used in these assays were recovered from starved
plates. Ideally, the behaviors should be assessed in animals that have
undergone the transient dauer stage, but this was impractical because of
complications from the pleiotropic developmental phenotypes. We also measured
the same behavioral responses in the ncr single and ncr-2; ncr-1;
daf-12 triple mutant adult animals grown under normal conditions, and
found that these mutants also had wild-type responses.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The molecular identities of the ncr and daf-9 genes
suggest a model in which NCR proteins are required for redistributing
internalized cholesterol to intracellular compartments, including the
endoplasmic reticulum (ER). The DAF-9 cytochrome P450 enzyme, which appears to
be ER localized (Gerisch et al.,
2001; Jia et al.,
2002
), may depend on sterol substrates delivered by the NCR
pathway for the biosynthesis of a DAF-12 ligand
(Gerisch and Antebi, 2004
;
Mak and Ruvkun, 2004
). In
ncr-2; ncr-1 mutants, defective sterol trafficking results in reduced
availability of DAF-9 substrates, thus recapitulating the pleiotropic
phenotypes of daf-9 and daf-12 class 6 (ligand binding
domain) mutants. This model predicts a suboptimal concentration of available
cholesterol in the ncr-2; ncr-1 mutant, but a normal or higher
available cholesterol concentration in the daf-9 mutant. Rescue of
ncr-2; ncr-1 by increased exogenous cholesterol is consistent with
this model.
Expression pattern of ncr genes
Two cDNA isoforms of ncr-1 were identified in this study. They
differ only in their 5'-UTR region caused by an alternative non-coding
first-exon located 8.7 kb upstream of the predicted coding region. A sequence
homologous to this non-coding exon is located 9.4 kb upstream of the predicted
C. briggsae ncr-1 gene in a similar arrangement, suggesting
functional significance. The expression pattern of the ncr-1 gene
reveals two interesting features. First, the ncr-1 gene expression
pattern largely coincides with the tissue distribution of cholesterol in
C. elegans (Matyash et al.,
2001; Merris et al.,
2003
), consistent with a function for NCR-1 in cholesterol
trafficking. Second, the hypodermal expression of the ncr-1 gene is
dynamically regulated by developmental and environmental signals. A similar
dynamic regulation of daf-9 expression has been reported
(Gerisch and Antebi, 2004
;
Gerisch et al., 2001
).
The expression of ncr-1 and ncr-2 colocalizes with
daf-9 and sdf-9 in a pair of neuroendocrine cells called XXX
cells (Ohkura et al., 2003).
In previous studies, ablating the XXX cells caused the formation of
characteristic partial dauers, suggesting that these cells are an important
source of the dauer-inhibiting hormone
(Ohkura et al., 2003
). It was
proposed that SDF-9 functions by increasing DAF-9 activity or helping to
execute DAF-9 function (Ohkura et al.,
2003
). We hypothesize that sdf-9 participates in a signal
transduction cascade that modulates the output of the daf-9 pathway
in concert with intracellular sterol levels. We found that sdf-9 is
strongly Dafc in combination with ncr-1, but not with ncr-2
mutations. This result can be explained by two alternative models. In the
first, NCR-1 and NCR-2 function redundantly in the XXX cells in cholesterol
trafficking, but NCR-1 plays a more important role than NCR-2. Alternatively,
NCR-1 is involved in cholesterol trafficking, whereas NCR-2 functions at a
different step in the dauer signaling pathway, perhaps parallel or downstream
to SDF-9 function. The broader expression of NCR-1, as well as its sensitivity
towards cholesterol deprivation and progesterone, suggests a major role in
cholesterol trafficking. However, further studies, such as determining the
subcellular localization of the NCR proteins, are necessary to help resolve
the two models.
The DAF-12 ligand
The current study supports the hypothesis that a hormone ligand for DAF-12
is derived from sterol. It has previously been shown that lack of DAF-9
function could not be bypassed by the addition of exogenous pregnenolone
(Jia et al., 2002), suggesting
that it is not the product of the DAF-9 enzyme. We find that pregnenolone
fails to alleviate the phenotypes of the ncr-2; ncr-1 mutant,
suggesting that pregnenolone or its derivatives are also unlikely to be the
substrates of the DAF-9 enzyme. Pregnenolone is the precursor of vertebrate
steroid hormones and the product of the first side-chain cleavage of
cholesterol. Consistent with our results, the C. elegans genome
contains no recognizable ortholog of mammalian cytochrome P450scc, which
catalyzes the complex multi-step reaction of pregnenolone synthesis
(Simpson, 1979
).
The insect molting hormone ecdysone is structurally different from
vertebrate steroid hormones. It contains the sterol multi-ring system and an
intact side chain, which is modified by oxidation (mostly hydroxylations) and
methylation at multiple positions (Gilbert
et al., 2002). As a result, ecdysone is amphipathic, a property
that helps its movement across cell boundaries and diffusion through tissues.
Although the pathway for ecdysone biosynthesis has not been fully
characterized, cytochrome P450 enzymes are likely to catalyze many of the
hydroxylation steps (Gilbert et al.,
2002
). Interestingly, it was recently reported that the levels of
hydroxycholesterols are diminished in cell lines lacking NPC1 function, and
among different mutant NPC1 lines, hydroxycholesterol levels correlate better
with the severity of cellular phenotypes than do ER cholesterol levels
(Frolov et al., 2003
). It is
possible that the hormonal ligand of DAF-12 is structurally more similar to
the insect hormone ecdysone, with an intact side chain and modified by
hydroxylations, than to the vertebrate steroid hormones.
C. elegans as a model system to study NPC1 function
Mammalian pathways for intracellular cholesterol trafficking are probably
conserved in C. elegans. In addition to ncr-1 and
ncr-2, the C. elegans genome contains an ortholog of the
human NPC2/HE1 gene, the locus responsible for the remaining 5% of cases of
NP-C disease (Friedland et al.,
2003; Ko et al.,
2003
; Naureckiene et al.,
2000
). The C. elegans genome also contains homologs of
LDL-receptor-like proteins (Grant and
Hirsh, 1999
; Ishihara et al.,
2002
; Yochem et al.,
1999
), caveolin (Scheel et
al., 1999
), START domain proteins, a large family of cytochrome
P450 enzymes (Menzel et al.,
2001
) and more than 250 nuclear hormone receptors
(Gissendanner et al.,
2004
).
Progesterone inhibits intracellular cholesterol trafficking in mammalian cell lines. We show that progesterone probably also inhibits cholesterol trafficking in C. elegans. First, progesterone treatment phenocopied the effects of cholesterol deprivation in wild-type animals; second, ncr-1 mutants were hypersensitive to progesterone treatment; and third, the effects of progesterone could be suppressed by cholesterol in the media. However, NCR proteins are unlikely to be the direct targets of progesterone, as progesterone treatment of either single mutant failed to phenocopy the ncr-2; ncr-1 double mutant.
In mammalian cells, intracellular trafficking of cholesterol and
sphingolipid are closely linked (Puri et
al., 1999; Zhang et al.,
2001
). Complex glycosphingolipids accumulate in NPC cells in
addition to cholesterol (Neufeld et al.,
1999
; Zhang et al.,
2001
). It has been hypothesized that the neurological symptoms of
NPC disease are caused by sphingolipid accumulation, because they accumulate
more extensively than cholesterol in the central nervous system, and because
the neuropathological changes in NPC disease resemble those in primary
sphingolipid storage disorders (Liu et
al., 2000
; Zervas et al.,
2001b
). Although little is known about the metabolism and
homeostasis of sphingolipids in C. elegans, we find neuronal
abnormalities in ncr-2; ncr-1 mutants similar to the ectopic
dendritogenesis observed in the mammalian NPC disease models, which is thought
to be caused by accumulation of the ganglioside GM2
(Walkley, 1998
;
Zervas et al., 2001a
).
In conclusion, C. elegans represents a useful model organism for studying conserved pathways of intracellular cholesterol trafficking. Classical genetic approaches and genome-wide screening methods can be employed to identify additional components that interact with the ncr genes and to yield potential therapeutic targets for the treatment of NP-C disease.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/22/5741/DC1
* Present address: Institute of Neuroscience, University of Oregon, Eugene,
OR 97403, USA
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agard, D. A., Hiraoka, Y., Shaw, P. and Sedat, J. W. (1989). Fluorescence microscopy in three dimensions. Methods Cell Biol. 30,353 -377.[Medline]
Ailion, M. and Thomas, J. H. (2000). Dauer
formation induced by high temperatures in Caenorhabditis elegans.Genetics 156,1047
-1067.
Antebi, A., Culotti, J. G. and Hedgecock, E. M.
(1998). daf-12 regulates developmental age and the dauer
alternative in Caenorhabditis elegans. Development
125,1191
-1205.
Antebi, A., Yeh, W. H., Tait, D., Hedgecock, E. M. and Riddle,
D. L. (2000). daf-12 encodes a nuclear receptor that
regulates the dauer diapause and developmental age in C. elegans.Genes Dev. 14,1512
-1527.
Bargmann, C. I. and Horvitz, H. R. (1991). Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans. Neuron 7, 729-742.[Medline]
Bargmann, C. I., Hartwieg, E. and Horvitz, H. R. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74,515 -527.[Medline]
Birnby, D. A., Link, E. M., Vowels, J. J., Tian, H., Colacurcio,
P. L. and Thomas, J. H. (2000). A transmembrane
guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of
chemosensory behaviors in Caenorhabditis elegans.Genetics 155,85
-104.
Blanchette-Mackie, E. J., Dwyer, N. K., Amende, L. M., Kruth, H. S., Butler, J. D., Sokol, J., Comly, M. E., Vanier, M. T., August, J. T., Brady, R. O. et al. (1988). Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes. Proc. Natl. Acad. Sci. USA 85,8022 -8026.[Abstract]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Butler, J. D., Blanchette-Mackie, J., Goldin, E., O'Neill, R.
R., Carstea, G., Roff, C. F., Patterson, M. C., Patel, S., Comly, M.
E., Cooney, A. et al. (1992). Progesterone blocks cholesterol
translocation from lysosomes. J. Biol. Chem.
267,23797
-23805.
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.
Cassada, R. C. and Russell, R. L. (1975). The dauer larva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol. 46,326 -342.[Medline]
Chitwood, D. J. (1999). Biochemistry and
function of nematode steroids. Crit. Rev. Biochem. Mol.
Biol. 34,273
-284.
Chun, K. T. and Simoni, R. D. (1992). The role
of the membrane domain in the regulated degradation of
3-hydroxy-3-methylglutaryl coenzyme A reductase. J. Biol.
Chem. 267,4236
-4246.
Coburn, C. M. and Bargmann, C. I. (1996). A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron 17,695 -706.[Medline]
Culotti, J. G. and Russell, R. L. (1978).
Osmotic avoidance defective mutants of the nematode Caenorhabditis
elegans. Genetics 90,243
-256.
Davies, J. P., Chen, F. W. and Ioannou, Y. A.
(2000). Transmembrane molecular pump activity of Niemann-Pick C1
protein. Science 290,2295
-2298.
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.
Frolov, A., Zielinski, S. E., Crowley, J. R., Dudley-Rucker, N.,
Schaffer, J. E. and Ory, D. S. (2003). NPC1 and NPC2
regulate cellular cholesterol homeostasis through generation of low density
lipoprotein cholesterol-derived oxysterols. J. Biol.
Chem. 278,25517
-25525.
Fujiwara, M., Ishihara, T. and Katsura, I.
(1999). A novel WD40 protein, CHE-2, acts cell-autonomously in
the formation of C. elegans sensory cilia.
Development 126,4839
-4848.
Gems, D., Sutton, A. J., Sundermeyer, M. L., Albert, P. S.,
King, K. V., Edgley, M. L., Larsen, P. L. and Riddle, D. L.
(1998). Two pleiotropic classes of daf-2 mutation affect
larval arrest, adult behavior, reproduction and longevity in
Caenorhabditis elegans. Genetics
150,129
-155.
Gerisch, B. and Antebi, A. (2004). Hormonal
signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer
diapause in response to environmental cues.
Development 131,1765
-1776.
Gerisch, B., Weitzel, C., Kober-Eisermann, C., Rottiers, V. and Antebi, A. (2001). A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span. Dev. Cell 1,841 -851.[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]
Gissendanner, C. R., Crossgrove, K., Kraus, K. A., Maina, C. V. and Sluder, A. E. (2004). Expression and function of conserved nuclear receptor genes in Caenorhabditis elegans. Dev. Biol. 266,399 -416.[CrossRef][Medline]
Grant, B. and Hirsh, D. (1999).
Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte.
Mol. Biol. Cell 10,4311
-4326.
Hedgecock, E. M., Culotti, J. G., Thomson, J. N. and Perkins, L. A. (1985). Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes. Dev. Biol. 111,158 -170.[Medline]
Hobert, O., Mori, I., Yamashita, Y., Honda, H., Ohshima, Y., Liu, Y. and Ruvkun, G. (1997). Regulation of interneuron function in the C. elegans thermoregulatory pathway by the ttx-3 LIM homeobox gene. Neuron 19,345 -357.[Medline]
Ioannou, Y. A. (2000). The structure and function of the Niemann-Pick C1 protein. Mol. Genet. Metab. 71,175 -181.[CrossRef][Medline]
Ishihara, T., Iino, Y., Mohri, A., Mori, I., Gengyo-Ando, K., Mitani, S. and Katsura, I. (2002). HEN-1, a secretory protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis elegans. Cell 109,639 -649.[CrossRef][Medline]
Jia, K., Albert, P. S. and Riddle, D. L.
(2002). DAF-9, a cytochrome P450 regulating C. elegans
larval development and adult longevity. Development
129,221
-231.
Jin, Y., Jorgensen, E., Hartwieg, E. and Horvitz, H. R.
(1999). The Caenorhabditis elegans gene unc-25
encodes glutamic acid decarboxylase and is required for synaptic transmission
but not synaptic development. J. Neurosci.
19,539
-548.
Kimura, K. D., Tissenbaum, H. A., Liu, Y. and Ruvkun, G.
(1997). daf-2, an insulin receptor-like gene that
regulates longevity and diapause in Caenorhabditis elegans.Science 277,942
-946.
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.
Komatsu, H., Mori, I., Rhee, J. S., Akaike, N. and Ohshima, Y. (1996). Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans.Neuron 17,707 -718.[Medline]
Kurzchalia, T. V. and Ward, S. (2003). Why do worms need cholesterol? Nat. Cell Biol. 5, 684-688.[CrossRef][Medline]
Li, W., Kennedy, S. G. and Ruvkun, G. (2003).
daf-28 encodes a C. elegans insulin superfamily member that
is regulated by environmental cues and acts in the DAF-2 signaling pathway.
Genes Dev. 17,844
-858.
Lin, K., Dorman, J. B., Rodan, A. and Kenyon, C.
(1997). daf-16: an HNF-3/forkhead family member that can
function to double the life-span of Caenorhabditis elegans.Science 278,1319
-1322.
Liscum, L. and Munn, N. J. (1999). Intracellular cholesterol transport. Biochim. Biophys. Acta 1438,19 -37.[Medline]
Liu, Y., Wu, Y. P., Wada, R., Neufeld, E. B., Mullin, K. A.,
Howard, A. C., Pentchev, P. G., Vanier, M. T., Suzuki, K. and Proia, R.
L. (2000). Alleviation of neuronal ganglioside storage does
not improve the clinical course of the Niemann-Pick C disease mouse.
Hum. Mol. Genet. 9,1087
-1092.
Lui, Z. and Ambros, V. (1991). Alternative temporal control systems for hypodermal cell differentiation in Caenorhabditis elegans. Nature 350,162 -165.[CrossRef]
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.
Mak, H. Y. and Ruvkun, G. (2004). Intercellular
signaling of reproductive development by the C. elegans DAF-9
cytochrome P450. Development
131,1777
-1786.
Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D.,
Thiele, C., Grant, B., Maxfield, F. R. and Kurzchalia, T. V.
(2001). Distribution and transport of cholesterol in
Caenorhabditis elegans. Mol. Biol. Cell
12,1725
-1736.
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Abstract]
Menzel, R., Bogaert, T. and Achazi, R. (2001). A systematic gene expression screen of Caenorhabditis elegans cytochrome P450 genes reveals CYP35 as strongly xenobiotic inducible. Arch. Biochem. Biophys. 395,158 -168.[CrossRef][Medline]
Merris, M., Wadsworth, W. G., Khamrai, U., Bittman, R.,
Chitwood, D. J. and Lenard, J. (2003). Sterol effects
and sites of sterol accumulation in Caenorhabditis elegans:
developmental requirement for 4alpha-methyl sterols. J. Lipid
Res. 44,172
-181.
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.
Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A. and Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389,994 -999.[CrossRef][Medline]
Ohkura, K., Suzuki, N., Ishihara, T. and Katsura, I.
(2003). SDF-9, a protein tyrosine phosphatase-like molecule,
regulates the L3/dauer developmental decision through hormonal signaling in
C. elegans. Development
130,3237
-3248.
Pentchev, P. G., Kruth, H. S., Comly, M. E., Butler, J. D.,
Vanier, M. T., Wenger, D. A. and Patel, S. (1986).
Type C Niemann-Pick disease. A parallel loss of regulatory responses in both
the uptake and esterification of low density lipoprotein-derived cholesterol
in cultured fibroblasts. J. Biol. Chem.
261,16775
-16780.
Puri, V., Watanabe, R., Dominguez, M., Sun, X., Wheatley, C. L., Marks, D. L. and Pagano, R. E. (1999). Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases. Nat. Cell Biol. 1, 386-388.[CrossRef][Medline]
Ren, P., Lim, C. S., Johnsen, R., Albert, P. S., Pilgrim, D. and
Riddle, D. L. (1996). Control of C. elegans
larval development by neuronal expression of a TGF-beta homolog.
Science 274,1389
-1391.
Riddle, D. L. and Albert, P. S. (1997). Regulation of dauer larva development. In C. elegans II (ed. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess), pp. 739-768. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Scalettar, B. A., Swedlow, J. R., Sedat, J. W. and Agard, D. A. (1996). Dispersion, aberration and deconvolution in multi-wavelength fluorescence images. J. Microsc. 182, 50-60.[Medline]
Schackwitz, W. S., Inoue, T. and Thomas, J. H. (1996). Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans. Neuron 17,719 -728.[Medline]
Scheel, J., Srinivasan, J., Honnert, U., Henske, A. and Kurzchalia, T. V. (1999). Involvement of caveolin-1 in meiotic cell-cycle progression in Caenorhabditis elegans. Nat. Cell Biol. 1,127 -129.[CrossRef][Medline]
Simpson, E. R. (1979). Cholesterol side-chain cleavage, cytochrome P450, and the control of steroidogenesis. Mol. Cell. Endocrinol. 13,213 -227.[CrossRef][Medline]
Sulston, J. and Hodgkin, J. (1988). Methods. InThe Nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 595. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Swoboda, P., Adler, H. T. and Thomas, J. H. (2000). The RFX-type transcription factor DAF-19 regulates sensory neuron cilium formation in C. elegans. Mol. Cell 5,411 -421.[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]
Thomas, J. H., Birnby, D. A. and Vowels, J. J.
(1993). Evidence for parallel processing of sensory information
controlling dauer formation in Caenorhabditis elegans.Genetics 134,1105
-1117.
Vanier, M. T. and Millat, G. (2003). Niemann-Pick disease type C. Clin. Genet. 64,269 -281.[CrossRef][Medline]
Vowels, J. J. and Thomas, J. H. (1992). Genetic
analysis of chemosensory control of dauer formation in Caenorhabditis
elegans. Genetics 130,105
-123.
Walkley, S. U. (1998). Cellular pathology of lysosomal storage disorders. Brain Pathol. 8, 175-193.[Medline]
Yochem, J., Tuck, S., Greenwald, I. and Han, M.
(1999). A gp330/megalin-related protein is required in the major
epidermis of Caenorhabditis elegans for completion of molting.
Development 126,597
-606.
Yu, S., Avery, L., Baude, E. and Garbers, D. L.
(1997). Guanylyl cyclase expression in specific sensory neurons:
a new family of chemosensory receptors. Proc. Natl. Acad. Sci.
USA 94,3384
-3387.
Zervas, M., Dobrenis, K. and Walkley, S. U. (2001a). Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations. J. Neuropathol. Exp. Neurol. 60, 49-64.[Medline]
Zervas, M., Somers, K. L., Thrall, M. A. and Walkley, S. U. (2001b). Critical role for glycosphingolipids in Niemann-Pick disease type C. Curr. Biol. 11,1283 -1287.[CrossRef][Medline]
Zhang, M., Dwyer, N. K., Neufeld, E. B., Love, D. C., Cooney,
A., Comly, M., Patel, S., Watari, H., Strauss, J. F., III, Pentchev, P.
G. et al. (2001). Sterol-modulated glycolipid sorting occurs
in niemann-pick C1 late endosomes. J. Biol. Chem.
276,3417
-3425.