From the a Lipid Cell Biology Section, Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the b Division of Neonatology, Department of Pediatrics, Georgetown University Medical Center, Washington, D. C. 20007, the c Neurobiology Research Laboratory, Veterans Affairs Medical Center, Newington, Connecticut 06111, the d Developmental and Metabolic Neurology Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892, e Tottori University, Faculty of Medicine, Yanago, Japan 683-8503, the f Saccomanno Research Institute, Saint Mary's Hospital, Grand Junction, Colorado 81502, theg Department of Pediatrics and Biological Structure, University of Washington, Seattle, Washington 98195, the h Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the i Department of Biochemistry and INSERM U 189, Faculte de Medecine, Lyon-Sud, Oullins, France 69921, and the j Department of Neurology, Mayo Clinic and Foundation, Rochester, Minnesota 55905
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking.
Immunocytochemical studies in fibroblasts reveal that the
NPC1 gene product resides in a novel set of
lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate
receptor() vesicles that can be distinguished from
cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol
transport out of lysosomes also redistribute NPC1 to cholesterol-laden
lysosomes. Sterol relocation from lysosomes in cultured human
fibroblasts can be blocked at 21 °C, consistent with
vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles
may transiently interact with lysosomes to facilitate sterol relocation.
Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein.
We conclude that a distinctive organelle containing NPC1 mediates
retrograde lysosomal transport of endocytosed cargo that is not
restricted to sterol.
Niemann-Pick C disease
(NP-C)1 is an
autosomal-recessive, fatal, neurodegenerative lysosomal storage
disorder (1). Human NPC1 (2) and its ortholog in the mouse
(3) have recently been identified by positional and complementation
cloning. NPC1 is predicted to be a polytopic membrane-spanning protein
with a domain of striking homology to the putative sterol-sensing
regions of other proteins that are essential for managing cellular
cholesterol homeostasis (2, 4). NPC1 also has a terminal di-leucine motif that targets proteins to late endocytic compartments (2).
The most recognized cellular lesion in NP-C cells is impaired lysosomal
relocation of endocytosed low density lipoprotein (LDL)-derived
cholesterol to other intracellular sites, such as the plasma membrane
and endoplasmic reticulum (5-7). Cholesterol accumulates in lysosomes
as well as the Golgi apparatus (8, 9). Induction of
cholesterol-mediated homeostatic responses is proposed to be delayed
because of deficient enrichment of regulatory sterol pools in the
endoplasmic reticulum (10, 11).
NP-C is also characterized by lysosomal accumulation of other
metabolites. Phospholipids, glycosphingolipids, sphingoid bases, cystine, and glycoproteins, as well as cholesterol, are stored in a
tissue-specific pattern (1, 12). To date, it has been assumed that the
transport defect in NP-C cells is specific to cholesterol and that
other metabolites accumulate secondarily to cholesterol. However, in
the brain, where functional impairment is progressive and ultimately
fatal, massive accumulation of glycolipids occurs, with little, if any,
excessive cholesterol storage (1, 12). Extensive glycolipid storage in
fetal human NP-C liver further suggests that this storage is an early
event in the development of the disorder (12). These glycolipid storage
anomalies do not readily integrate into a unified pathogenic mechanism
involving a primary defect in sterol trafficking. The possibility that
the NP-C lesion might alternatively reflect a more generalized defect in trafficking out of lysosomes has not been previously explored.
Here we provide evidence that sterol transport from lysosomes is
vesicle-mediated, as suggested by a previous report (13). We show that
NPC1 resides in a novel set of vesicles that appear to interact with
cholesterol-filled lysosomes. We further show that mutations in NPC1 do
not exclusively affect sterol transport, but rather, target a
vesicle-mediated retrograde transport pathway, potentially affecting a
wide range of endocytosed material. Cholesterol, in addition to being a
cargo, appears to modulate NPC1 vesicle-mediated transport from lysosomes.
Materials--
Fetal bovine serum was obtained from HyClone
Laboratories, Inc., Logan, UT. Lipoprotein-deficient bovine serum
(LPDS) and human LDL were prepared by Intracel Corp. (Rockville, MD).
Glass and plastic microscope culture wells (Lab-Tek) were purchased from Thomas Scientific. [14C(U)]Sucrose (442 mCi/mM) was
purchased from NEN Life Science Products. Filipin was purchased from
Polysciences (Warrington, PA). Progesterone, purchased from Sigma, and
U18666A (3- Tissue Culture--
Normal and NP-C fibroblasts were derived
from volunteers and confirmed patients of the Developmental and
Metabolic Neurology Branch under the guidelines approved by the NINDS
Intramural Review Board. Five different normal cell lines (ENZ123,
ENZ125, ENZ143, GM5565, and GM1652) and three NP-C cell lines (GM3123,
DMN92.31, and DMN87.57) were used. A null mutant NP-C cell line
(DMN98.16), negative for NPC1 mRNA and protein
expression,2 was used solely
in immunocytochemical studies.
Fibroblasts were cultured in Eagle's minimal essential medium
supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mM glutamine, and 100 units of
penicillin/streptomycin/ml in humidified 95% air and 5%
CO2 at 37 °C. For biochemical analyses, fibroblasts
seeded at a density of 80,000 cells/well in plastic 6-well dishes
(Costar, Cambridge, MA) were incubated for 5-7 days in McCoy's medium
with 5% LPDS supplemented as above. For immunocytochemical analyses,
fibroblasts were seeded at a density of 20,000 cells/well in
McCoy's/5% LPDS medium in 9.5-cm glass microscope wells (Nunc, Inc.,
Naperville, IL) coated with human fibronectin.
Biochemical Measurement of Fluid-phase
Endocytosis--
Fluid-phase endocytosis was measured using
[14C]sucrose as described (14-16). Briefly, cells were
washed once with McCoy's/5% LPDS medium and then incubated for 3 h at 37 °C in prewarmed medium containing 5 µCi of
[14C]sucrose (specific activity, 11,000 dpm/pmol). The
labeling medium was removed, and the cells were washed six times with
ice-cold serum-free McCoy's medium. For reflux experiments, cells were recultured in medium at 37 °C. At specified times, the medium was
transferred to tubes and centrifuged at 4 °C for 5 min at 1500 rpm
(to pellet any cells present). Cell monolayers were washed with cold
serum-free McCoy's medium and then solubilized in 1% SDS for 30 min.
Total cell protein was determined by the method of Lowry et
al. (17). [14C]Sucrose activity was assayed by
counting aliquots of the medium and the solubilized cell monolayers in
a scintillation counter (Tri-Carb, model 4000; United Technologies
Packard, Downer's Grove, IL). The percentage of
[14C]sucrose in cells = ((cell-associated
[14C]sucrose)/(cell-associated [14C]sucrose + medium [14C]sucrose)) × 100.
Kinetic Data Analysis--
[14C]Sucrose data were
analyzed using WinSAAM, the Windows version of the SAAM (Simulation,
Analysis and Modeling) program (18-20) and a four-compartment model.
Compartments 1-3 represent intracellular pools of
[14C]sucrose that turn over at distinct rates;
compartment 4 represents the culture medium. This is a modified version
of the model proposed by Blomhoff et al. (21), which
contained compartments that turned over in 10, 18, and 330 min and were
considered to represent early, late and terminal (lysosomal) endocytic
compartments, respectively. Because only a few sampling times were
available before 30 min, we retained the first compartment in the model
but fixed its turnover to be 10 min. Loss of tracer from compartments 1 and 2 is directed to the medium, with no assumptions made about the
pathway of delivery involved. Based on our immunocytochemical findings
(see under "Results"), we directed loss from compartment 3 (lysosomes) back into compartment 2 (late endosomes) rather than into
the medium.
Pilot studies indicated that [14C]sucrose loading for
3 h provided optimal conditions for egress studies. The model was
set up to simulate the loading period so as to determine the
distribution of [14C]sucrose among the intracellular
compartments by the end of loading. To simulate cellular clearance, the
value for compartment 4 (culture medium) was set to zero, and the
amount of tracer remaining in compartments 1, 2, and 3 (expressed as a
percentage of dose in cells at the end of loading) was calculated for
24 h. Calculated values were compared with the observed data.
Transfer rates for pathways, other than loss from compartment 1, were
adjusted iteratively by the software until the calculated curves fit
each set of experimental data.
Calculations--
Data from each study (normal cell line and
NP-C cell line) were fit simultaneously by the model. Differences were
introduced in specific pathways until the model fit both the normal and
NP-C data (or normal ± LDL data). The model was also used to
predict the pool sizes at steady state. The latter were calculated by simulating a constant input into compartment 1 for each study. The
values for the pool sizes were expressed relative to a mass of 100% in
compartment 1 of normal cells.
Statistics--
Because the normal and NP-C cell lines were
studied in pairs, differences were tested using the paired t
test. Differences in transfer rates between normal and NP-C cells were
expressed as a percentage of normal in each study (rather than the
absolute value because this varied with each cell line).
NPC1 Antipeptide Antibodies--
Polyclonal antibodies were
raised against keyhole limpet hemocyanin-conjugated synthetic peptides
representing residues 75-93 (NPC1-L), 147-165 (NPC1-N), and
1256-1274 (NPC1-C) of the human NPC1 protein (Fig. 1). The peptides
were synthesized on an automated Symphony/Multiplex peptide synthesizer
using HBYU/NMP protocol (FMOC chemistry) and purified by reverse-phase
high performance liquid chromatography. The sequences were verified by
amino acid analysis, analytical high pressure liquid chromatography,
and electrospray mass spectrometry. New Zealand White rabbits were injected subcutaneously with 1 mg of keyhole limpet
hemocyanin-conjugated peptide (in 0.5 ml of phosphate-buffered saline)
mixed thoroughly into an emulsion with 0.5 ml of Freund's complete
adjuvant. For subsequent boosts, keyhole limpet hemocyanin-conjugated
peptide (0.5 mg in Freund's incomplete adjuvant) was injected
subcutaneously 3 and 6 weeks later. Sera obtained from these animals
contained polyclonal antibodies against NPC1 protein. Western blot
analysis of human fibroblast cell lines reveals that all three NPC1
antipeptide antisera identify a 165-kDa
protein.3
Immunocytochemical Analyses--
Cells in glass chamber slides
were washed in phosphate-buffered saline and fixed in 3%
paraformaldehyde for 30 min. Cells were immunolabeled using an indirect
procedure in which all incubations (quench, primary and secondary
antibodies, and washes) were performed in blocker solution containing
filipin (0.05%) and goat IgG (2.5 mg/ml). Primary antibodies used were
NPC1 (1:1000), LAMP2 (1:40), and MPR (1:1000). Secondary fluorescein
isothiocyanate- and LRSC-labeled antibodies were used at 1:50 dilution.
Fluorescence was viewed with a Zeiss laser scanning confocal microscope
using a krypton-argon Omnichrome laser with excitation wavelengths of
488 and 568 nm for fluorescein isothiocyanate and LRSC, respectively.
Filipin fluorescence was viewed using an argon ion laser (Coherent)
with an excitation wavelength of 351 nm.
NPC1 Protein Resides in a Distinct Vesicular Compartment--
We
established the intracellular localization of NPC1 in cultured human
fibroblasts using antibodies against three distinct 19-amino acid
peptide sequences of the protein (Fig.
1). Although all three antisera
immunostained similar structures, NPC1-C antiserum was routinely used
because it provided the brightest staining. Wild type and some mutated
forms of NPC1 localized in granular structures (Fig.
2, A and B). NPC1
immunostaining is specific because it is blocked by co-incubation with
NPC1-C peptide and is absent in "null mutant" NP-C fibroblasts that
do not express NPC1 (Fig. 2C).
To further characterize the cellular structures containing NPC1, we
compared its distribution to LAMP2, a marker for both late endosomes
and lysosomes (22), in normal fibroblasts enriched with LDL cholesterol
(Fig. 3). NPC1 extensively colocalizes
with LAMP2 (Fig. 3A). However, NPC1 is found in some, but
not all, LAMP2(+) vesicles. Thus, it appears that a subset of LAMP2(+) vesicles contains NPC1. NPC1(+) vesicles are not enriched by either endocytosed LDL-derived cholesterol (Fig. 3B), a lysosomal
marker (8), or endocytosed DiIC16(3), a lipophilic
fluorescent dye (23) that colocalizes with endocytosed LDL-derived
cholesterol (data not shown). NPC1(+) vesicles in some NP-C fibroblast
lines (such as GM3123) also do not become enriched with endocytosed cholesterol and can be clearly distinguished from cholesterol-laden LAMP2(+) lysosomes typical of such mutant cells (data not shown). NPC1(+) vesicles are also marked by antibodies to lysosomal integral membrane protein I, but not by antibodies to adaptins and cathepsin (data not shown). MPR, a specific marker for late endosomes (24), does
not localize in NPC1(+) vesicles (Fig.
4). Thus, NPC1 appears to reside in a
novel NPC1(+)/LAMP2(+) vesicle that is neither a MPR(+)-late endosome
nor a cholesterol-enriched lysosome.
Drugs That Block Transport of Cholesterol out of Lysosomes Induce
Lysosomal Sequestration of NPC1 Protein--
Because the documented
mutations of NPC1 indicate that it plays a critical role in
transporting endocytosed cholesterol from lysosomes, one might expect
that this protein can interact with cholesterol-enriched lysosomes. We
tested whether drugs such as U18666A (25) and progesterone (26), which
block cholesterol transport out of lysosomes, might also alter the
cellular distribution of NPC1. Cytochemical analysis reveals that in
addition to the sequestration of cholesterol in lysosomes (25, 26),
U18666A (Fig. 5) and progesterone (not
shown) also trap the majority of NPC1 protein within these same
organelles. When progesterone is washed out of cells at 37 °C, to
re-establish cholesterol transport (26), distinct NPC1(+)/LAMP2(+) and
NPC1 ( Cholesterol Transport From Lysosomes Is
Temperature-dependent--
The localization of NPC1 to
LAMP2(+) vesicles and its drug-induced relocation to cholesterol-filled
lysosomes suggest that sterol transport from lysosomes is
vesicle-mediated. To further evaluate this potential mode of
trafficking, progesterone was again used to reversibly accumulate
LDL-derived cholesterol in lysosomes (7, 26). During the subsequent
progesterone washout phase, the temperature was reduced to determine
whether lysosomal cholesterol relocation could be blocked at
temperatures that perturb vesicular trafficking (27). Cytochemical
analysis reveals that the pool of cholesterol sequestered in lysosomes
(Fig. 6A) was readily
mobilized during progesterone washout at 37 °C (Fig. 6B) but not at 21 °C (Fig. 6C). In similar experiments, the
relocation of lysosomal [3H]cholesterol to the plasma
membrane was also blocked below 21 °C (Fig.
7). Taken together, these results suggest
that NPC1 participates in a vesicle-mediated transport of cholesterol
from lysosomes.
Clearance of Endocytosed [14C]Sucrose Is Impaired in
NP-C Fibroblasts--
Because a vesicle-mediated mechanism of sterol
transport from lysosomes allows potential cotransport of additional
lysosomal cargo, we examined whether NP-C cells are also defective in
clearing other endocytosed material. [14C]Sucrose, a
fluid-phase marker for vesicular transport (14-16), was used to
monitor endocytic trafficking. We tested whether mutated NPC1 alters
vesicular transport in the absence of lysosomal cholesterol storage.
Lysosomes of normal and NP-C fibroblasts were cleared of sterol by
extended incubation in lipoprotein-deficient medium prior to loading
with [14C]sucrose (8) and then were maintained in such
medium during subsequent [14C]sucrose loading and
clearance. As illustrated in Fig. 8,
clearance of endocytosed [14C]sucrose was delayed in all
NP-C cell lines examined. The difference in clearance was apparent at
30 min and was maintained thereafter.
Compartmental Modeling Predicts That NP-C Fibroblasts Are Defective
in Clearance of [14C]Sucrose from a Late Endocytic
Compartment--
In order to identify the cellular compartments
through which sucrose transport might be altered by a nonfunctional
NPC1 protein, we constructed a compartmental model for the endocytic
uptake and release of tracer [14C]sucrose in normal and
NP-C fibroblasts (Fig. 9). In normal
fibroblasts, the relative accumulation of endocytosed cargo (Table
I) and the calculated turnover times of
compartments 1, 2, and, 3 (10 min, 1 h, and
In NP-C fibroblasts, the calculated rates of [14C]sucrose
efflux from intracellular compartments 1 and 3 were identical to those for normal cells, but the rate from compartment 2 was retarded (47% of
normal, as shown in Fig. 9 for egress data displayed in Fig. 8).
Release from compartment 2 into the medium was significantly lower in
NP-C cells compared with normal cells (0.71 ± 0.23/h versus 0.44 ± 0.17/h (mean ± S.D.),
respectively; p < 0.01 for eight paired experiments).
Thus, the rate of transport from compartment 2 into the medium is
nearly one-half the rate seen in normal cells (62.6 ± 14.4%;
n = 8). Uptake into compartment 1 from the medium was
3.63E-5 ± 0.8E-5/h in normal versus 4.35E-5 ± 0.9E-5/h in NP-C fibroblasts (n = 8). Movement into
compartment 3 from compartment 2 (0.54 ± 0.16/h) and release from
compartment 3 (0.05 ± 0.01/h) were the same in both normal and
NP-C cells. The modeling reveals that the impaired movement from late
endosomes back into the medium accounts for the observed delayed
clearance of [14C]sucrose from NP-C cells (as depicted in
Fig. 8).
The compartmental modeling also predicts the NP-C phenotype, namely,
excessive lysosomal storage of endocytosed cargo (see Table I). Most
(88%) of the excess endocytosed cargo retained in the three NP-C
fibroblast lines studied is predicted to accumulate in compartment 3 (lysosomes). The [14C]sucrose mass predicted to
accumulate in lysosomes of NP-C cells is about 2-fold greater than
normal cells, consistent with the previously reported 2-fold increase
in the fluid-phase volume (29, 30) and endocytosed sterol content (6,
7, 10) of NP-C lysosomes.
Cellular Cholesterol Enrichment Retards Clearance of
Endocytosed [14C]Sucrose from Normal Fibroblasts--
We
explored the possibility that endocytosed cholesterol is not only a
cargo of the NPC1-mediated retrograde lysosomal transport pathway but
may also modulate this pathway through the sterol-sensing domain of the
NPC1 protein. Lipoprotein-depleted normal fibroblasts were incubated in
the absence or presence of LDL during both the endocytic uptake and
subsequent clearance of [14C]sucrose. As seen in Fig.
10, cellular sterol enrichment
significantly retarded [14C]sucrose clearance in normal
fibroblasts.
In order to identify the cellular compartments potentially targeted by
this cellular sterol enrichment, we applied the same compartmental
model (Fig. 9). Sterol enrichment significantly reduced
[14C]sucrose release from compartment 2 back into the
medium (0.71 ± 0.23/h versus 0.57 ± 0.21/h,
p < 0.05; for (-)LDL versus (+)LDL, respectively) in three different normal fibroblast lines. Consistent with a recent study (31), cellular sterol enrichment did not
alter the rate of trafficking of bulk flow markers between early
endocytic compartments and the cell surface.
The well documented lesions in lysosomal sterol processing that
occur in Niemann-Pick C disease have established a role for NPC1 in
sterol transport out of lysosomes. The identification of putative
sterol-sensing domains in the recently cloned NPC1 gene
further links this protein to cellular sterol processing (2). Our
present studies reveal that in cultured human fibroblasts, NPC1
identifies a novel vesicle that appears to interact with cholesterol-filled lysosomes. We provide evidence that NPC1 mediates a
vesicular form of transport out of lysosomes and that the cargo transported along this pathway is not restricted to sterol.
We have shown that NPC1 protein (Fig. 2) resides in a novel set of
vesicles that are LAMP2(+) (Fig. 3) and MPR( Several independent lines of research have provided evidence for
retrograde lysosomal transport. Lysosomal membrane proteins appear to
continuously cycle between lysosomes and the plasma membrane via
endosomal compartments (32-39). Endocytosed fluid-phase cargo also
transfers from lysosomes back to late endocytic compartments (40, 41).
Physical interactions between MPR(+)-late endosomes and lysosomes that
allow content mixing have been reported (41-46). The NPC1(+)
cholesterol-laden lysosome induced by drugs in the current studies
(Fig. 5) could represent a hybrid organelle analogous to that which
forms from the fusion of a late endosome with a lysosome (44, 45).
Drugs that retard cholesterol transport from lysosomes may allow fusion
of NPC1 vesicles with lysosomes but block their subsequent fission. The
observed reappearance of NPC1 vesicles as well as presumed lysosomes
cleared of sterol after removal of progesterone from cell cultures
(data not shown) is consistent with a reversible mechanism of drug
interdiction involving blocked fission of a hybrid organelle.
NP-C fibroblasts were found to be defective in vesicle-mediated
retrograde transport of [14C]sucrose in the absence of
lysosomal cholesterol storage (Fig. 8). Compartmental modeling was used
to identify the kinetic compartment associated with this intracellular
trafficking defect. The modeling targets the defect in retrograde
transport to a late endocytic compartment (Fig. 9) and predicts that
this defect is primarily responsible for the lysosomal storage (Table
I) seen in NP-C cells. The extent of the predicted defect in the rate
of [14C]sucrose transport from the late endocytic
compartment to the cell surface is comparable to the reported defect in
endocytosed sterol transport from lysosomes to the cell surface in NP-C
fibroblasts (approximately 50%) (6, 7). The kinetically defined late endocytic compartment likely represents the immunocytochemically defined NPC1 vesicles.
The role that endocytosed sterol plays in NP-C disease may be quite
different than previously envisioned. The defect in vesicle-mediated egress of both [14C]sucrose and cholesterol suggests that
the NPC1-mediated transport pathway is not restricted to sterol
mobilization. Defective cellular clearance of a fluid-phase marker in
the absence of lysosomal cholesterol storage in NP-C suggests that
generalized, bulk retrograde transport of vesicular components is
impaired. Several studies have provided evidence that endocytosed
lipids (47) and proteins (48) recycle to the cell surface from late
endocytic compartments. Increasingly, attention has focused on the
sorting and trafficking of endocytosed plasma membrane glycolipids
(49). The defect in retrograde vesicular transport that we presently
report could account for the excessive GM2 accumulation
seen in endosomal compartments of some NP-C cell lines (50). Recently
we demonstrated that endocytosed GM2 is a specific marker
for the NPC1 vesicle,4 consistent with a role for this
organelle in the intracellular trafficking of both glycolipids and
cholesterol. Disruption of glycolipid transport might contribute to the
cellular pathology associated with NP-C. Glycolipids, such as
GM2, accumulate extensively in the absence of excessive
cholesterol storage in NP-C neurons (51, 52) and in the whole brain (1,
12). Interestingly, glycolipids and cholesterol are the major lipid
components of membrane microdomains found in rafts;
detergent-insoluble, glycolipid-enriched complexes; and caveolae (49).
Preliminary cellular extraction studies show that NPC1 has the
solubility properties of a raft-associated protein.5 Future studies will
clarify the possible structural and functional relationships that NPC1
and lipid microdomains might share.
We also provide preliminary evidence that endocytosed sterol itself may
modulate functions of NPC1. Cellular sterol enrichment retards the
clearance of endocytosed [14C]sucrose from normal
fibroblasts (Fig. 10) and, like NPC1 mutations, specifically targets a
late endocytic compartment (Fig. 9). Thus, in addition to being cargo,
endocytosed sterol appears to alter the rate of vesicular trafficking
along this pathway, possibly interacting with the sterol-sensing domain
of NPC1.
In conclusion, the present findings suggest that a novel NPC1 organelle
mediates retrograde lysosomal trafficking of a potentially wide
spectrum of lysosomal cargo. The tissue-specific pattern of metabolites
that accumulate in NP-C disease likely reflects the diverse cargo that
is transported along this pathway. In the liver, where endocytosed
lipoprotein processing can be significant, sterol may represent a major
portion of the endocytosed cargo utilizing this pathway. Alternatively,
in neurons and other types of cells in which little sterol may be
exogenously derived, NPC1 may predominantly regulate retroendocytic
glycolipid trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-(2-(diethylamino))ethoxy)androst-5-en-17-one), generously supplied by Dr. W. Andrus (The Upjohn Co.), were stored as 1 and 10 mg/ml stock solutions, respectively, in ethanol at -20 °C.
Mouse anti-human lysosome-associated membrane protein-2 (LAMP2) and
lysosomal integral membrane protein I antibodies, developed by Dr.
J. T. August, were obtained from the Developmental Studies
Hybridoma Bank maintained by the University of Iowa (Iowa City, IA).
Monoclonal anti-
-adaptin (AP-1) and anti-
-adaptin (AP-2)
antibodies were obtained from Sigma. Mouse anti-human cathepsin D
antibodies were obtained from Chemicon International, Inc. (Temecula, CA). Antibodies to the 300-kDa cation-independent mannose 6-phosphate receptor (MPR) were a gift of Dr. Suzanne Pfeffer. Fluorescein isothiocyanate- and LRSC-labeled secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
NPC1 antipeptide antibodies. The NPC1
protein (2) contains as many as 16 transmembrane domains (black
stripes). The "sterol-sensing" region of NPC1 is contained
within transmembrane domains 4-8 (boxed). The carboxyl
terminus contains a di-leucine motif (LLNF). The location of the
peptide sequences within the predicted domains of the NPC1 protein used
to generate polyclonal antibodies are shown. NPC1-L, peptide
within the leucine zipper region (striped box; residues
73-94) of the unique NPC1 amino-terminal region (residues 55-165);
NPC1-N, a different peptide within the unique NPC1
amino-terminal region; NPC1-C, peptide within the
carboxyl-terminal tail.
View larger version (102K):
[in a new window]
Fig. 2.
Intracellular distribution of NPC1.
Normal (A), mutant NP-C (GM3123) (B), and null
NP-C (DMN98.16) (C) human fibroblasts were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days and then incubated in
fresh medium containing LDL (50 µg/ml) for 24 h to enrich
cellular membranes with sterol. Fibroblasts were immunostained for NPC1
as described under "Experimental Procedures." Note the robust
staining of granular structures in normal (A) and mutant
(B) NPC1 fibroblasts and the absence of cellular staining in
the NP-C fibroblast line (C) that does not express
NPC1.
View larger version (45K):
[in a new window]
Fig. 3.
NPC1 colocalizes with LAMP2 but not with
endocytosed cholesterol. Normal human fibroblasts were incubated
in McCoy's/5% LPDS medium at 37 °C for 4 days and then incubated
in fresh medium containing LDL (50 µg/ml) for 24 h to enrich
cellular membranes with sterol. Fibroblasts were immunostained for NPC1
(A-D; green) and LAMP2 (A and C;
red) and cytochemically stained with filipin to reveal the
cellular distribution of endocytosed cholesterol (B and
D; blue), as described under "Experimental
Procedures." Confocal microscopy revealed that although NPC1 always
colocalized with LAMP2 (A; yellow), many LAMP2(+)
vesicles do not contain NPC1 (A; red). The
LAMP2(+)/NPC1( ) vesicles (A; red) are lysosomes
enriched with endocytosed LDL cholesterol (B;
blue). Note that the NPC1(+)/LAMP2(+) vesicles
(A; yellow), however, do not become enriched with
endocytosed LDL cholesterol (B; green). The
region in the rectangle in A is shown at higher
magnification in C and D. Whereas the
NPC1(
)/LAMP2(+) lysosomes (C; red) become
enriched with endocytosed cholesterol (D; blue), the
NPC1(+)/LAMP2(+) vesicles (C; yellow) do not
(D; green).
View larger version (24K):
[in a new window]
Fig. 4.
NPC1 does not colocalize with mannose
6-phosphate receptor. Normal human fibroblasts were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days and then incubated in
fresh medium containing LDL (50 µg/ml) for 24 h to enrich
cellular membranes with sterol. Fibroblasts were immunostained for MPR
(green) and NPC1 (red). Note that the
intracellular distribution of NPC1 is quite different from that of MPR,
with little apparent colocalization.
)/LAMP2(+) vesicles cleared of cholesterol are formed (data not
shown). These observations suggest that a normally transient
interaction of NPC1 with cholesterol-laden lysosomes is required to
relocate cholesterol to other cellular sites.
View larger version (44K):
[in a new window]
Fig. 5.
U18666A induces NPC1 to colocalize with
endocytosed cholesterol in lysosomes. Normal human fibroblasts
were incubated in McCoy's/5% LPDS medium at 37 °C for 4 days,
washed in phosphate-buffered saline, and then incubated in fresh medium
containing LDL (50 µg/ml) and U18666A (2 µg/ml) for 24 h to
allow endocytosed LDL-derived cholesterol to accumulate in lysosomes.
Cells were immunostained for NPC1 (red) and cytochemically
stained with filipin to reveal cholesterol (green). The
diagonal line marks the position at which the cell was
scanned in the x-z axis. A portion of the x-z
scan (bracketed by the arrows) is shown in B-D.
A merge (B) of the NPC1 image (red, as seen in
C) and the cholesterol image (green, as seen in
D) shows extensive yellow color, indicating that
the two are largely colocalized.
View larger version (63K):
[in a new window]
Fig. 6.
Cytochemical assessment of lysosomal
clearance of LDL-derived cholesterol at 21 °C. Normal human
fibroblasts were incubated in McCoy's/5% LPDS medium at 37 °C for
4 days to deplete sterols from cellular membranes. A, cells
were then incubated in fresh medium containing LDL (50 µg/ml) and
progesterone (10 µg/ml) for 24 h to load lysosomes with
cholesterol. A portion of the cultures was washed in phosphate-buffered
saline and then incubated in McCoy's/5% LPDS medium for 24 h at
either 37 °C (B) or 21 °C (C). Confocal
microscopy revealed abundant perinuclear lysosomal filipin cholesterol
fluorescence in cells following endocytic LDL loading (A),
which cleared 24 h after incubation in LPDS-containing medium at
37 °C (B) but not at 21 °C (C).
View larger version (15K):
[in a new window]
Fig. 7.
Effect of temperature on mobilization of
lysosomal cholesterol to the plasma membrane in cultured
fibroblasts. Normal human fibroblasts (2.0 × 106) were seeded in 75-cm2 flasks in
McCoy's/5% LPDS medium for 4 days. Cells were subsequently incubated
in fresh medium with 20 µg/ml [3H]cholesteryl linoleate
LDL (3.0 × 107dpm/mg protein) for 24 h. Cultures
were washed with McCoy's medium (4×) and then incubated for 6 h
at the indicated temperatures. Cell extracts were prepared and
fractionated in 10% Percoll gradients as described (7). High density
-hexosaminidase-enriched fractions were designated as lysosomes, and
low density alkaline phosphatase-enriched fractions were designated as
plasma membrane. A notable block in transfer of endocytosed cholesterol
from lysosomes to the plasma membrane was seen below 21 °C, the
temperature at which vesicular transport is blocked. Note that delayed
progesterone washout at low temperatures cannot account for the
observed reduction in relocation of lysosomal
[3H]cholesterol. The half-life of cellular
[3H]progesterone washout is 40 min at 37 °C and less
than twice that at 16 °C.
View larger version (12K):
[in a new window]
Fig. 8.
The effect of NPC1 mutations on the clearance
of endocytosed [14C]sucrose from cholesterol-depleted
cells. Normal and NP-C human fibroblast cell lines were incubated
in McCoy's/5% LPDS medium at 37 °C for 5-7 days to clear
endocytosed cholesterol from lysosomes. Cells were incubated in
McCoy's/5% LPDS medium containing 5 µCi/ml
[14C]sucrose for 3 h at 37 °C to load endocytic
compartments with the tracer, washed at 4 °C, and then incubated in
McCoy's/5% LPDS medium at 37 °C for the indicated times.
[14C]Sucrose associated with the cell monolayer and
medium was measured as described. Each data point represents
the mean value ± S.D. of triplicate culture wells. The graph
represents the results of a single representative paired experiment.
Data points are shown as squares for normal fibroblasts
(GM5565) and as diamonds for NP-C fibroblasts (GM3123). The
calculated fits to the data, using the model shown in Fig. 9, are
represented by solid and dashed curves for normal
and NP-C fibroblasts, respectively. The model is available at
htp://biomodel.georgetown.edu/model.
24 h, respectively)
correspond to previously reported values for early endocytic, late
endocytic, and lysosomal compartments (15, 16, 21, 28).
View larger version (12K):
[in a new window]
Fig. 9.
Model for cellular uptake and release of
[14C]sucrose in fibroblasts. Circles
represent compartments, numbers identify compartments, and
arrows represent movement between compartments. Compartment
4 represents the extracellular medium, and compartments 1-3 are
intracellular. Values above the arrows represent the
fraction of compartment transferred to an adjacent compartment per
hour, for a representative experiment. The initial conditions in
compartment 4 at the start of the loading was 8 × 106
dpm, and uptake into compartment 1 was 4.7E-5/h and 3.5E-5/h, in normal
and NP-C fibroblasts, respectively. The turnover time is represented by
the reciprocal of the sum of the losses from each compartment,
i.e. the turnover time of compartment 2 in normal
fibroblasts = 1/(0.612/h + 0.459/h) = 56 min. Data and model
calculated fits for cellular clearance in this representative study are
shown in Fig. 8 (normal versus NP-C) and Fig. 10 (normal
versus normal + LDL).
Predicted percentage of accumulation of internalized
[14C]sucrose in endocytic compartments of cultured
fibroblasts
View larger version (11K):
[in a new window]
Fig. 10.
The effect of endocytosed LDL on the
cellular clearance of endocytosed [14C]sucrose.
Normal human fibroblast cell lines were incubated in McCoy's/5% LPDS
medium at 37 °C for 5-7 days to deplete membrane sterol. Cells were
incubated in McCoy's/5% LPDS medium in the absence or presence of LDL
(50 µg/ml) for 24 h and then in McCoy's/5% LPDS medium
containing 5 µCi/ml [14C]sucrose in the absence or
presence of LDL (50 µg/ml) for 3 h at 37 °C to load endocytic
compartments with the tracer. Cells were washed and then incubated in
McCoy's/5% LPDS medium in the absence or presence of LDL (50 µg/ml)
at 37 °C for the indicated times. [14C]Sucrose
associated with the cell monolayer and medium was measured as
described. Each data point represents the mean ± S.D.
of triplicate culture wells. The graph represents the results of a
single representative paired experiment using normal fibroblasts
(GM5565). Data points are shown as squares and
diamonds for cells in the absence and presence of LDL,
respectively. The calculated fits to the data, using the model shown in
Fig. 9, are represented by solid and dashed
curves for cells in the absence and presence of LDL,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (Fig. 4) and that do
not accumulate endocytosed lysosomal markers (Fig. 3). The ganglioside
GM2 exclusively localizes to NPC1(+) vesicles and not to
lysosomes containing endocytosed cholesterol, confirming their unique
identity.4 We found that
drugs that block sterol transport out of lysosomes (U18666A and
progesterone) also trap NPC1 in cholesterol-laden lysosomes (Fig. 5).
Sterol relocation from lysosomes can be blocked by low temperature
(Figs. 6 and 7) and drugs (13) that affect vesicular trafficking. Taken
together, these findings suggest that NPC1 vesicles transiently
interact with lysosomes to transfer sterol to other cellular sites.
Definitive characterization of the novel NPC1(+) vesicular compartment
as well as other organelles that may participate in retrograde
lysosomal transport is currently under investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Yiannis A. Ioannou for both helpful discussions in the early phase of this project and critical reading of the manuscript, Dr. Jean Wilson for critical reading of the manuscript, and Devera Schoenberg for help in the preparation of the manuscript. We are grateful to the Ara Parseghian Medical Research Foundation and the National Niemann Pick Disease Foundation for their encouragement.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
k To whom correspondence should be addressed: Lipid Cell Biol. Section, Bldg. 8, Rm. 427, NIDDK, National Institutes of Health, 8 Center Dr., MSC 0850, Bethesda, MD 20892. Tel.: 301-496-2050; Fax: 301-402-0723; E-mail: joanbm{at}bdg8.niddk.nih.gov.
2 E. B. Neufeld, M. Wastney, S. Patel, S. Suresh, A. M. Cooney, N. K. Dwyer, C. F. Roff, K. Ohno, J. A. Morris, E. D. Carstea, J. P. Incardona, J. F. Strauss III, M. T. Vanier, M. C. Patterson, R. O. Brady, P. G. Pentchev, and E. J. Blanchette-Mackie, unpublished data.
3 S. Patel, manuscript in preparation.
4 E. J. Blanchette-Mackie, manuscript in preparation.
5 J. Incardona, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NP-C, Niemann-Pick C
disease;
GM2, GalNAc1-4[NeuAc
2-3]Gal
1-4Glc
1-Cer;
LAMP, lysosome-associated membrane protein;
LDL, low density lipoprotein;
LPDS, lipoprotein-deficient serum;
MPR, mannose 6-phosphate receptor;
NPC1, Niemann-Pick C1 protein;
LRSC, lissamine rhodamine sulfonyl
chloride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|