From the Canadian Institutes of Health Research Group
on the Molecular and Cell Biology of Lipids and the Departments of
§ Medicine,
Biochemistry, and
§§ Cell Biology, University of Alberta,
Edmonton, Alberta T6G 2S2, Canada
Received for publication, May 31, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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Niemann Pick type C (NPC) disease is a
progressive neurodegenerative disorder. In cells lacking functional
NPC1 protein, endocytosed cholesterol accumulates in late
endosomes/lysosomes. We utilized primary neuronal cultures in which
cell bodies and distal axons reside in separate compartments to
investigate the requirement of NPC1 protein for transport of
cholesterol from cell bodies to distal axons. We have recently observed
that in NPC1-deficient neurons compared with wild-type neurons,
cholesterol accumulates in cell bodies but is reduced in distal axons
(Karten, B., Vance, D. E., Campenot, R. B., and Vance,
J. E. (2002) J. Neurochem. 83, 1154-1163). We
now show that NPC1 protein is expressed in both cell bodies and distal
axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies
from low density lipoproteins (LDLs), high density lipoproteins, or
cyclodextrin complexes was transported into axons in normal amounts,
whereas transport of endogenously synthesized cholesterol was impaired.
Inhibition of cholesterol synthesis with pravastatin in wild-type and
NPC1-deficient neurons reduced axonal growth. However, LDLs restored a
normal rate of growth to wild-type but not NPC1-deficient neurons
treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does
not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the
neurological dysfunction in NPC disease.
Niemann Pick Type C
(NPC)1 disease is a fatal
disorder characterized by progressive neurodegeneration in the central
nervous system and accumulation of lipids in peripheral tissues
(reviewed in Refs. 1 and 2). NPC1, the gene responsible for
95% of cases of NPC disease, encodes the 1278-amino acid transmembrane NPC1 protein (3) that resides mainly in a subset of late endosomes but
also transiently associates with lysosomes and the
trans-Golgi network (4, 5). In
Balb/cNctr-npcN/N mice, a murine model of NPC
disease, NPC1 protein is not expressed because of a deletion/insertion
mutation in the npc1 gene (6). These mice are asymptomatic
at birth but exhibit tremor and ataxia and die prematurely at ~3
months. In both humans and mice, NPC1 deficiency results in a
progressive loss of neurons in the cerebellum (7).
Cells with dysfunctional NPC1 accumulate unesterified cholesterol in
late endosomes/lysosomes (8-11). In addition, homeostatic responses to
exogenously supplied cholesterol, for example down-regulation of
cholesterol biosynthesis and low density lipoprotein (LDL) receptor
expression, and activation of cholesterol esterification, are blunted
(11, 12). These defects have been ascribed to an impaired egress of
endocytosed LDL-derived cholesterol from lysosomes (11, 13). More
recent evidence suggests that the intracellular trafficking of all
cholesterol that transits the plasma membrane is affected in cells
lacking functional NPC1 (14, 15). NPC1 is therefore thought to play a
role in transporting cholesterol out of lysosomes/late endosomes. The
conclusion that NPC1 plays a key role in cholesterol trafficking and
homeostasis is consistent with the observation that the protein
contains a sequence that is homologous to a putative sterol-sensing
domain identified in two other proteins involved in cholesterol
homeostasis, namely 3-hydroxy-3-methylglutaryl-CoA reductase and SCAP
(sterol response element-binding protein
cleavage-activating protein) (16,
17). Recently, NPC1 has also been shown to contain regions of homology
to the RND family of prokaryotic permeases and has been proposed to
mediate fatty acid transport (18).
The intracellular itinerary of endocytosed LDL cholesterol involves
passage into late endosomes where cholesteryl esters are hydrolyzed,
and the released unesterified cholesterol is subsequently distributed
to the endoplasmic reticulum, Golgi apparatus, and plasma membrane (19,
20). The internalization of LDLs and the ensuing hydrolysis of
cholesteryl esters are normal in NPC1-deficient cells (12). Upon exit
from sorting late endosomes, cholesterol appears to travel initially to
the plasma membrane and subsequently to NPC1-containing late endosomes.
In the absence of NPC1, cholesterol accumulates in
multivesicular/multilamellar vesicles (9).
Vesicular trafficking of endocytosed material has been investigated
extensively in nonpolarized cells such as fibroblasts (reviewed in
Refs. 21 and 22). In neurons, as in other polarized cells, an
additional targeting step is required for delivery of protein cargo
specifically to axonal or dendritic membranes (corresponding to apical
or basolateral domains, respectively, of other polarized cells).
Generally, this targeting is mediated by the trans-Golgi network (reviewed in Ref. 23). The transport of lipids has received less attention. Several studies indicate that membrane lipids are
inserted into axonal membranes in the vicinity of their final destination (24, 25) and that lateral diffusion of lipids within the
axonal membrane is too slow to provide sufficient membrane materials
for axonal growth (26).
Unlike the major membrane phospholipid, phosphatidylcholine, which is
synthesized in both cell bodies and distal axons of sympathetic neurons
(27-29), cholesterol is synthesized only in cell bodies and must be
transported into axons for assembly into their plasma membranes (28).
Newly made cholesterol is transported from the endoplasmic reticulum to
the plasma membrane by a rapid, energy-dependent mechanism
that bypasses the Golgi apparatus (30, 31). Cultured sympathetic
neurons do not require exogenously supplied cholesterol for growth but
can obtain all the cholesterol they need from endogenous synthesis
(32). Similarly, cholesterol in the brain is derived mainly from
endogenous synthesis (33-35). However, Mahley (36) has proposed that
after peripheral nerve injury, cholesterol is salvaged from
degenerating neurons and myelin and reutilized for axonal regeneration
in a process involving endocytosis of apoE-containing lipoproteins
secreted by endoneurial macrophages (37). In NPC1-deficient mice, this
pathway appears to be defective (38). In the central nervous system,
cholesterol is thought to be recycled by a similar process during axon
remodeling. Although most plasma lipoproteins do not cross the
blood-brain barrier, cerebrospinal fluid contains lipoprotein particles
(reviewed in Refs. 39-41), and some apolipoproteins (apoE, apoD, and
apoJ) are expressed in the brain (42-44). In addition, several LDL
receptor family members are expressed in the central nervous system
(reviewed in Ref. 45).
In the present study, we have studied the metabolism and transport of
cholesterol in compartmented cultures of sympathetic neurons (46) from
wild-type and NPC1-deficient mice. We show that NPC1 is expressed in
both cell bodies and distal axons and that the amount of LDL-derived
cholesterol that reaches distal axons is the same in wild-type and
NPC1-deficient neurons. However, the transport of endogenously made
cholesterol is impaired. Moreover, LDL-derived cholesterol does not
support axonal growth of NPC1-deficient neurons when endogenous
cholesterol biosynthesis is inhibited.
Materials--
L15 medium was purchased from Invitrogen. Other
cell culture material was from BD Biosciences (Bedford, MA). Mouse 2.5 S nerve growth factor (NGF) was purchased from Alomone Laboratories
Ltd. (Jerusalem, Israel). Rat serum was provided by the University of
Alberta Animal Services and was delipidated by the method of Cham and
Knowles (47). Teflon dividers used for construction of compartmented
cultures were purchased from Tyler Research Instruments (Edmonton,
Canada). [1a,2a(n)-3H]cholesteryl oleate (44 Ci/mmol),
[7(n)-3H]cholesterol (5 Ci/mmol), and
[1-14C]acetic acid (57 mCi/mmol) were obtained from
Amersham Biosciences (Baie d'Urfé, Canada). Pravastatin was a
gift from Dr. S. Yokoyama (Nagoya City University, Nagoya, Japan).
U18666A was purchased from Biomol Research Laboratories (Plymouth
Meeting, PA). Progesterone, cholesterol, cholesteryl oleate, and
methyl-
A rabbit anti-human NPC1 polyclonal antibody (raised against amino
acids 1261-1272 of NPC1) was used for immunoblotting and was a
generous gift from Dr. D. Ory (Washington University, St. Louis, MO).
The rabbit anti-mouse polyclonal NPC1 antibody used for
immunocytochemical studies was raised against amino acids 1254-1273 of
NPC1 and was provided by Dr. W. S. Garver (University of Arizona,
Tucson, AZ). The rat anti-mouse LAMP1 antibody was from PharMingen
(Lexington, KY). Alexa Fluor 488-labeled goat anti-rabbit IgG and Texas
Red-labeled goat anti-rat IgG were from Molecular Probes (Eugene, OR).
All other materials were from Sigma
Primary Cultures of Sympathetic Neurons--
Superior cervical
ganglia were dissected from 1-day-old mouse pups from a breeding colony
of Balb/cNctr-npcN/+ mice established
at the University of Alberta from original breeding pairs obtained from
Jackson Laboratories (Bar Harbor, ME). The mice were maintained under
temperature-controlled conditions with a 12-h light:12-h dark cycle and
supplied with food and water ad libitum. Breeders were fed a
9% fat breeder diet (Purina LabDiet, Richmond, IN). Henceforth, mice
homozygous or heterozygous for the npc1 mutation will be
referred to as npc
Compartmented culture dishes were assembled as described previously
(50). A Teflon divider was sealed to the dish with silicon grease,
partitioning the dish into three separate compartments. Dissociated
neurons were plated in the center compartment. Within 2-3 days axons
elongated along tracks, crossing under the silicon grease barrier into
the left- and right-side compartments. The center compartment thus
contained cell bodies with proximal axons, whereas the side
compartments contained pure distal axons. No exchange of fluid occurs
between compartments (50). In some experiments the culture dish was
divided into five compartments with a Teflon divider. Neurons were
plated in the center compartment, and axons extended right and left
sequentially into the two adjacent compartments.
Immunoblotting for NPC1 Protein--
Cell bodies/proximal axons
and distal axons of wild-type murine sympathetic neurons were harvested
separately into ice-cold homogenization buffer (20 mM
Tris/HCl (pH 7.4), 1 mM EDTA, 0.25 mM sucrose)
containing 1 mM phenylmethylsulfonyl fluoride and a
protease inhibitor mixture (Complete Mini; Roche Molecular
Biochemicals). The samples were homogenized, transferred to
microcentrifuge tubes, and centrifuged at 4 °C for 10 min at
1,500 × g to pellet nuclei and unbroken cells. The
supernatant was centrifuged in a Beckmann TLA100.2 rotor at
436,000 × g for 30 min at 4 °C. The membrane pellet
was resuspended in buffer containing 50 mM Tris/HCl (pH 7.4), 0.15 M NaCl, 2 mM EDTA, and 1% Triton
X-100. The proteins were resolved by electrophoresis on 6%
polyacrylamide gels containing 0.1% SDS under reducing conditions and
then transferred to nitrocellulose. NPC1 expression was analyzed by
immunoblotting using a rabbit polyclonal antibody raised against a
peptide of human NPC1 (dilution 1:1,000). Immunoreactive proteins were
detected by reaction with peroxidase-conjugated goat anti-rabbit IgG
(dilution 1:10,000) and visualized with ECL reagent (ECL Western
blotting system; Amersham Biosciences).
Immunocytochemistry and Confocal Microscopy--
Neurons in
compartmented dishes were washed twice with phosphate-buffered saline
(PBS), to which was added 0.4% methylcellulose to reduce shear stress
and prevent neurons from detaching from the dish. The cells were fixed
for 20 min at room temperature in 4% paraformaldehyde and then
incubated in PBS containing 10% goat serum and 50 µg/ml saponin for
2 h at room temperature. For immunocytochemical localization of
LAMP1 and NPC1, the primary antibodies were rat anti-mouse LAMP1
(dilution 1:800) and rabbit anti-mouse NPC1 (dilution 1:500),
respectively. Secondary antibodies were Texas Red-labeled goat anti-rat
IgG (dilution 1:200 for LAMP1) and Alexa Fluor 488-labeled goat
anti-rabbit IgG (dilution 1:200 for NPC1). Pictures were taken using a
Zeiss LSM 510 confocal laser scanning microscope (Jena, Germany).
Excitation wavelengths were 488 nm (Alexa Fluor 488) and 543 nm (Texas Red).
Lipoprotein Isolation and Labeling--
Human LDLs and HDLs were
isolated from normolipemic volunteers by ultracentrifugation of plasma
in a KBr gradient as described (51). ApoE-free HDLs, which were
isolated after passage over a heparin-Sepharose column (52), were
generously provided by Dr. G. Francis (University of Alberta). All of
the lipoprotein concentrations are given as mg protein/ml. The
lipoproteins were labeled with [1a,2a-3H]cholesteryl
oleate (53). Briefly, 100 µCi of [3H]cholesteryl oleate
and 100 mg of phosphatidylcholine were dried under a stream of
nitrogen, and then 500 ml of PBS (pH 7.4) were added. The tube was
shaken gently at room temperature for 2 min and then sonicated on ice
under nitrogen for 10 min. Next, 500 ml of PBS (pH 7.4), 500 ml of
human lipoprotein-deficient serum, and 500 ml of LDLs or apoE-free HDLs
(~3 mg protein/ml) were added, and the mixture was incubated under
nitrogen for 18 h at 37 °C. The labeled lipoproteins were
isolated by ultracentrifugation (51). Specific radioactivities of
lipoproteins were 50-100 dpm/ng protein for [3H]LDLs and
80-120 dpm/ng protein for [3H]HDLs.
Preparation of Radiolabeled Cholesterol-Cyclodextrin
Complex--
Radiolabeled cholesterol was incorporated into
cyclodextrin complexes as described (54). Briefly, 0.98 mmol of
unlabeled cholesterol and 100 µCi of [3H]cholesterol
were dried under nitrogen. A solution of 13.4 mg of M Measurement of Axonal Extension--
Distal axons were removed
mechanically from the side compartments with a jet of sterile distilled
water delivered with a syringe through a 22-gauge needle (a process
termed axotomy) (55, 56). Axonal extension was measured using a Nikon
Diaphot inverted microscope equipped with a MD2 microscope digitizer
(Accustage Corp. Minneapolis, MN), which tracks stage movements to an
accuracy of ± 0.005 mm. In each culture, 16 tracks were measured
for each side compartment.
Anterograde Transport of Cholesterol into Distal Axons--
Cell
bodies/proximal axons of 12-day-old compartmented cultures were
incubated with 50 µl/dish of LDLs containing
[3H]cholesteryl oleate (0.1 mg protein/ml) or HDLs
containing [3H]cholesteryl oleate (0.19 mg protein/ml) or
0.25 mM M Statistical Analyses--
The statistical significance of
differences (p < 0.05) was determined using the
Student's t test.
Expression of NPC1 Protein in Murine Sympathetic
Neurons--
Sympathetic neurons from npc+/+,
npc+/
The distribution of NPC1 protein between cell bodies/proximal axons and
distal axons of murine sympathetic neurons was examined by
immunoblotting proteins in membrane-enriched fractions using an
anti-NPC1 antibody. Fig. 1 shows that
NPC1 is present in cell bodies/proximal axons as well as distal axons.
The immunoreactive band corresponded to a protein of ~170 kDa,
consistent with the size of glycosylated NPC1 reported in fibroblasts,
astrocytes, and brain homogenates (57-59). This band was absent from
npc
Immunocytochemical analysis of wild-type murine sympathetic neurons
using confocal microscopy revealed the presence of NPC1 in a punctate
distribution in cell bodies (Fig.
2A) and axons (Fig.
2D). The NPC1-positive compartments appeared to be smaller in size than those containing the lysosomal protein LAMP1, which was
also present both in cell bodies (Fig. 2B) and axons (Fig. 2E). A similar difference in size between NPC1- and
LAMP1-positive entities has previously been noted in mouse fibroblasts
(57). Phase contrast micrographs are shown in panels C and
F of Fig. 2.
Transport of Cholesterol from Cell Bodies to Axons--
A
characteristic of cells lacking functional NPC1 is an impaired egress
of LDL-derived cholesterol from late endosomes/lysosomes and a delayed
arrival of cholesterol at the plasma membrane (11, 13, 60). Henderson
et al. (61) have reported a defect in metabolism of
LDL-derived cholesterol in NPC1-deficient striatal neurons. However, no
detailed studies have been reported on the transport of cholesterol
transport into axons of NPC1-deficient neurons. We therefore used
compartmented neuron cultures (46) to investigate whether or not the
transport of endocytosed LDL-derived cholesterol from cell bodies to
distal axons of sympathetic neurons depended on functional NPC1. Cell
bodies/proximal axons were incubated with LDLs containing
[3H]cholesteryl ester, after which the distribution of
[3H]cholesterol between cell bodies/proximal axons and
distal axons was determined. As a measure of anterograde cholesterol
transport, the amount of unesterified [3H]cholesterol in
distal axons was calculated as a percentage of unesterified
[3H]cholesterol in cell bodies/proximal axons plus distal
axons. Because lysosomes and late endosomes are thought to be
restricted to neuronal cell bodies (62) and because the exit of
cholesterol from these compartments is thought to be defective in
NPC1-deficient cells, we expected that the transport of LDL-derived
cholesterol into distal axons would be defective in
npc
LDL uptake by cell bodies/proximal axons, calculated as the total
radioactivity in cholesterol plus cholesteryl ester in cell bodies and
axons divided by the specific radioactivity (dpm/ng protein) of LDLs,
was normal in npc
To test whether the [3H]cholesterol in distal axons was
derived from internalization of LDLs by the proximal axons in the
center compartment, we grew wild-type neurons in five-compartment
dishes in which cell bodies/proximal axons reside in the center
compartment, intermediate axons are in the two adjacent compartments,
and distal axons are in the two compartments farthest from the cell
bodies. When LDLs containing [3H]cholesteryl ester were
added to intermediate axons alone, no [3H]cholesterol was
detected in distal axons, indicating that processing of LDL cholesteryl
esters in the cell bodies is required for anterograde transport into
distal axons.
To confirm that LDL-derived cholesterol does not bypass the endocytic
pathway (i.e. is not taken up independently of the LDL receptor) in sympathetic neurons, we performed analogous experiments with sympathetic neurons from LDL receptor-null mice. After 44 h,
the extent of hydrolysis of cholesteryl esters was markedly lower in
LDL receptor-deficient neurons (11.5 ± 1.5%) than in wild-type
neurons (79.9 ± 5.1%), and the anterograde transport of
LDL-derived [3H]cholesterol in LDL receptor-deficient
neurons was negligible. These data confirm that in wild-type neurons
the uptake of LDL-[3H]cholesterol is largely mediated via
LDL receptors.
We also investigated whether or not the anterograde transport of three
other pools of cholesterol was impaired in neurons lacking functional
NPC1. These three pools of cholesterol were newly synthesized
cholesterol (labeled by [14C]acetate), a cytoplasmic pool
(derived from apoE-free HDLs), and a pool delivered to the plasma
membrane via cyclodextrin (M Progesterone and U18666A Differentially Affect Cholesterol
Transport in Neurons--
Because progesterone and the amphiphilic
amine U18666A have been reported to induce an NPC-like phenotype
(i.e. the accumulation of cholesterol-laden vesicles) in
fibroblasts and Chinese hamster ovary cells incubated with LDLs (63,
64), we examined cholesterol transport in wild-type sympathetic neurons
treated with U18666A or progesterone. We have recently demonstrated
that when sympathetic neurons are incubated with LDLs and either
U18666A or progesterone, cholesterol accumulates in cell bodies (49).
Fig. 4 shows that progesterone did not
significantly reduce the transport of LDL-derived cholesterol,
HDL-derived cholesterol, or endogenously synthesized cholesterol into
distal axons. In contrast, U18666A inhibited the anterograde transport
of cholesterol derived from both HDLs and LDLs, as well as endogenously
made cholesterol, into distal axons (Fig. 4). Thus, although neither
NPC1 deficiency (Fig. 3) nor progesterone disrupted the anterograde
axonal transport of lipoprotein-derived cholesterol, U18666A did
inhibit this process.
Because U18666A has been reported to inhibit cholesterol biosynthesis
at the level of squalene epoxidase (65), we confirmed that U18666A also
inhibits cholesterol biosynthesis in sympathetic neurons. Sympathetic
neurons were incubated with U18666A and [14C]acetate for
44 h. The incorporation of radiolabel into cholesterol in
U18666A-treated cells was only 33.3 ± 3.7% (3 independent
experiments) of that in neurons incubated without U18666A. In contrast,
progesterone did not inhibit cholesterol biosynthesis; the
incorporation of [14C]acetate into cholesterol in
progesterone-treated cells was the same (100.4 ± 12.1%, three
independent experiments) as in cells incubated without progesterone.
These data demonstrate that in neurons U18666A does not mimic the
effect of either progesterone or NPC1 deficiency on the anterograde
transport of LDL cholesterol.
A Defect in LDL Metabolism Becomes Apparent in npc
Because cholesteryl esters from apoE-free HDLs are believed to be taken
up by cells via a nonendocytic mechanism (67), we hypothesized that HDL
cholesterol might support normal axonal growth of pravastatin-treated
npc Utilization of Cholesterol for Axonal Growth of Neurons Treated
with U18666A or Progesterone--
We next measured axonal growth in
experiments similar to those depicted in Fig. 5 except that wild-type
neurons were treated with progesterone or U18666A. When the neurons
were incubated with U18666A, axonal extension was inhibited by
44.2 ± 3.8% after 6 days (Fig.
6A), and LDLs did not rescue
the impaired growth (Fig. 6A). The extent of inhibition of
growth was similar to that induced by pravastatin (39.7 ± 3.1%;
Fig. 5A). However, in contrast to pravastatin-treated
wild-type neurons, the addition of LDLs to U18666A-treated neurons did
not restore normal growth (Fig. 5A). Progesterone, on the
other hand, did not inhibit axonal extension (Fig. 6B),
although addition of pravastatin to progesterone-treated neurons (Fig.
6B) inhibited axonal growth to an extent similar to that
induced by pravastatin alone (compare Figs. 5A and
6B). Moreover, LDLs restored normal growth to neurons
incubated with progesterone + pravastatin (Fig. 6B).
Because U18666A partially (by 25-30%) inhibited cholesteryl ester
hydrolysis (calculated as radioactivity in
[3H]cholesterol as a percentage of total radioactivity in
cholesterol plus cholesteryl esters), the failure of LDL to rescue
axonal extension of U18666A-treated neurons might have been due to a limited availability of unesterified cholesterol derived from LDL
cholesteryl esters. To investigate this possibility, we assessed the
ability of various concentrations of LDLs to rescue the impaired axonal
extension induced by pravastatin. As shown in Fig. 6C, even
when we added only 50% of the LDLs used routinely to
pravastatin-treated neurons, a normal rate of axonal extension was
restored. Thus, it is unlikely that the 25-30% reduction in
cholesteryl ester hydrolysis by U18666A was the reason why inhibition
of axonal extension by U18666A was not overcome by addition of LDL.
Thus, progesterone and U18666A differentially affect cholesterol
metabolism in sympathetic neurons, and neither compound phenocopies
NPC1 deficiency. Table I summarizes these
observations.
We have investigated the anterograde transport of cholesterol in
sympathetic neurons from a murine model of NPC disease. Although neurons of the central nervous system are primarily affected in this
disease, we selected sympathetic neurons as our model system because
these neurons can be grown in compartmented cultures that are amenable
to studying transport of lipids and proteins between cell bodies and
axons, as well as axonal growth (46, 56). The major findings are: (i)
distal axons of wild-type sympathetic neurons contain NPC1 protein as
well as the lysosomal protein, LAMP1; (ii) the amount of LDL-derived
cholesterol transported from cell bodies to distal axons is normal in
npc Transport of Cholesterol from Cell Bodies to Distal
Axons--
We compared the transport of cholesterol from cell
bodies to distal axons in neurons of the three npc genotypes
using cholesterol synthesized endogenously from
[14C]acetate, as well as cholesterol derived from
exogenously added [3H]cholesteryl ester-labeled
lipoproteins and [3H]cholesterol complexed to
cyclodextrin. The anterograde transport of lipoprotein-derived
cholesterol and M
These data raise the question of how LDL-derived cholesterol that has
been added to cell bodies reaches distal axons without functional NPC1.
Two alternative pathways might mediate this anterograde transport. One
possibility is that LDL-derived cholesterol is transported into axons
without entering late endosomes. This, however, seems unlikely because
LDL cholesteryl esters are hydrolyzed at a normal rate in
npc
Cruz et al. have proposed a model for cholesterol
trafficking in Chinese hamster ovary cells in which endocytosed
LDL-derived cholesterol arrives rapidly at the plasma membrane,
regardless of the presence of NPC1, and is subsequently internalized
into NPC1-containing sorting endosomes (69). A lack of NPC1 was
proposed to delay the export of cholesterol from this sorting
compartment (69). Such an itinerary for LDL cholesterol would seemingly be consistent with our observation that normal amounts of LDL cholesterol are transported into distal axons of
npc Supply of Cholesterol for Axonal Growth--
Our laboratory has
previously demonstrated that inhibition of cholesterol synthesis in rat
sympathetic neurons inhibits axonal growth and that delivery of
cholesterol from LDLs restores normal growth (32, 72). In accordance
with these findings we now show that axonal growth of neurons of the
three npc genotypes is also reduced when cholesterol
biosynthesis is inhibited in the absence of an exogenous source of
cholesterol. We were thus able to establish whether or not exogenously
added cholesterol could be used for axonal growth of
npc Comparison of Phenotypes Induced by NPC1 Deficiency, Progesterone,
and U18666A in Sympathetic Neurons--
Our experiments demonstrate
that U18666A, progesterone, and NPC1 deficiency produce overlapping, as
well as distinct, modifications of cholesterol metabolism and transport
in neurons. As in fibroblasts and Chinese hamster ovary cells (9, 63,
64), these drugs induce an accumulation of cholesterol-filled vesicles
in the cytoplasm of neurons, similar to the situation in NPC1
deficiency (49). However, neither U18666A nor progesterone mimicks the
growth and transport defects occurring in
npc
We speculate that inhibition of cholesterol synthesis by U18666A might,
at least partially, be responsible for the defect in cholesterol
transport (Fig. 4) and axonal growth (Fig. 6) induced by U18666A (65).
A recent study by Fan et al. (73) showed that when
cortical neurons were incubated with inhibitors of cholesterol synthesis (i.e. compactin, which inhibits
hydroxymethylglutaryl-CoA reductase, or TU-2078, which inhibits
squalene epoxidase), the microtubule-associated protein Tau was
hyperphosphorylated, and depolymerization of microtubules occurred.
These observations might explain why inhibition of cholesterol
synthesis impairs cholesterol transport in neurons. However, the
mechanism by which U18666A inhibits axonal growth is likely more
complex than inhibition of cholesterol synthesis alone because LDLs
restore normal growth to neurons treated with pravastatin alone or
progesterone with pravastatin but not to neurons incubated with
U18666A. In this respect, U18666A mimicks NPC1 deficiency. These
observations suggest that in npc Conclusions--
Our experiments show that LDL cholesterol is
transported into axons of npc
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin (M
CD) were purchased from Sigma. Silica gel
G60 thin layer chromatography plates were from Merck. Polyacrylamide
gel electrophoresis and immunoblotting supplies were obtained from
Bio-Rad.
/
and
npc+/
, respectively, whereas
wild-type mice will be termed npc+/+. Because
npc
/
mice do not produce offspring,
npc+/
mice were used for breeding. Ganglia of
each mouse pup were placed separately into sterile microcentrifuge
tubes containing L15 medium supplemented with 10% rat serum and 50 ng/ml NGF. Ganglia were kept overnight at 4 °C but for no longer
than 24 h prior to dissociation, during which time npc
genotype was determined from tail clippings by PCR analysis of genomic
DNA using primers described previously (6). This protocol allowed
pooling of ganglia of a single genotype. Neuron cultures were plated
either on 48-well plates at a density of 1 ganglion/well or into the
center slot of compartmented culture dishes at a density of 0.6 ganglia/dish (48, 49). Medium in side compartments contained 100 ng/ml
NGF, whereas the center compartment contained 2.5% rat serum. During
the first week, 10 mM cytosine arabinoside and 20 ng/ml NGF
were included in the center compartment. Typically, the neurons were
cultured for 10-12 days prior to the start of an experiment. Breeding
pairs of LDL receptor-null mice (stock number 00064) were purchased
from Jackson Laboratories, and a colony of these mice was established
at the University of Alberta.
CD in 2 ml of
L15 medium was added and then sonicated for 10 min on ice and incubated
for 15 h at 37 °C. The solution contained 5 mM
M
CD/cholesterol with a M
CD:cholesterol molar ratio of 10:1 and a
specific radioactivity of 10 mCi/mmol M
CD. Directly before use, the
solution was filter-sterilized to remove microcrystals of cholesterol
not complexed with M
CD, resulting in a final specific radioactivity
of 6.6 mCi/mmol M
CD.
CD/[3H]cholesterol complex, and
[14C]acetate (10 mCi/ml) to label cholesterol synthesized
endogenously in cell bodies, in L15 medium containing 2.5% delipidated
rat serum. Lipoprotein concentrations were standardized so that
equivalent amounts of cholesterol were applied from LDLs and HDLs.
After 44 h (24 h for [3H]cholesterol/M
CD), all of
the compartments were washed twice with PBS containing 0.4%
methylcellulose and then once with PBS. The dishes were placed on ice,
and cell bodies/proximal axons and distal axons were harvested
separately into PBS containing 0.1% deoxycholate and 1 mg/ml EDTA. An
aliquot was used for measurement of protein using the BCA protein assay
(Pierce). The lipids were extracted from the remainder of the sample
with hexane/isopropanol 3:2 (v/v) and separated by thin layer
chromatography in the solvent system hexane/diisopropyl ether/acetic
acid (65:35:3 v/v/v), with authentic unesterified cholesterol and
cholesteryl ester standards added as carriers. Cholesterol and
cholesteryl ester were visualized with iodine vapor. Anterograde
transport of cholesterol is expressed as the amount of radiolabeled
unesterified cholesterol in distal axons as a percentage of
radiolabeled unesterified cholesterol in cell bodies/proximal axons + distal axons. To ensure that no other complex lipids co-migrated with
unesterified cholesterol, we included a saponification step for one set
of cell lysates labeled with [14C]acetate. For this
purpose, lipid extracts were incubated with 3% KOH in methanol (w/v)
for 30 min at 60 °C. The lipids were extracted twice with hexane and
then separated by thin layer chromatography. Recovery of radioactivity
in unesterified cholesterol of the saponified sample was 97.2 ± 9.7% of that in the nonsaponified sample. Radioactivity in cholesteryl
ester was negligible under both conditions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and npc
/
mice grow at the same rate, are similarly responsive to NGF, and
exhibit the same morphology when viewed by light microscopy (49). We
have shown that although npc+/+,
npc+/
and npc
/
sympathetic neurons as a whole contain equal amounts of cholesterol, the partitioning of cholesterol between cell bodies and axons is
altered in npc
/
neurons such that
unesterified cholesterol accumulates in cell bodies but is reduced in
distal axons (49).
/
sympathetic neurons.
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Fig. 1.
Expression of NPC1 protein in distal axons
and cell bodies/proximal axons of murine sympathetic neurons.
Murine sympathetic neurons were cultured for 12 days in compartmented
culture dishes. Cell bodies/proximal axons and distal axons were
harvested separately. Membrane-enriched fractions were prepared from
7.5 dishes combined (47 µg of protein from distal axons and 65 µg
of protein from cell bodies/proximal axons), and the proteins were
separated by electrophoresis on 6% polyacrylamide gels containing
0.1% SDS. The proteins were analyzed by immunoblotting using
antibodies directed against NPC1. The experiment was performed four
times with similar results.
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[in a new window]
Fig. 2.
Immunocytochemical localization of NPC1
protein in neurons. The expression of NPC1 and LAMP1 was
visualized by immunocytochemistry and confocal microscopy in cell
bodies (A and B) and distal axons (D
and E). A and D, NPC1. B
and E, LAMP1. C and F, Phase contrast
micrographs of cell bodies and distal axons, respectively. The
scale bars represent 20 µm. The experiment was performed
four times with similar results.
/
neurons. This, however, was not the
case; the same amount of LDL-[3H]cholesterol was
transported into distal axons of npc+/+,
npc+/
, and npc
/
neurons (Fig. 3). No
[3H]cholesteryl ester was detected in distal axons.
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Fig. 3.
Anterograde transport of cholesterol in
sympathetic neurons. Cell bodies/proximal axons of 12-day-old
compartmented cultures of sympathetic neurons were incubated in medium
containing delipidated rat serum with three different sources of
cholesterol: [3H]cholesteryl oleate-labeled LDLs (0.1 mg
of protein/ml), [3H]cholesteryl oleate-labeled apoE-free
HDLs (0.19 mg of protein/ml), or 0.25 mM
M CD/[3H]cholesterol complex. In all experiments,
endogenously synthesized cholesterol (Endo) was labeled with
10 µCi/ml [14C]acetate. After 44 h (24 h for
M
CD), cell bodies/proximal axons and distal axons were harvested
separately. Five dishes were combined for each sample of
npc+/+ (filled bars),
npc+/
(shaded bars), and
npc
/
(open bars) neurons.
Cellular lipids were extracted and separated by thin layer
chromatography, and radioactivity was measured in unesterified
cholesterol. The data are the means ± S.E. of four independent
experiments for LDL, HDL, and endogenous cholesterol and one experiment
for M
CD cholesterol. The data are expressed as the amounts of
radiolabeled unesterified cholesterol in distal axons as percentages of
radioactivity in cholesterol in cell bodies/proximal axons + distal
axons. *, p < 0.025 compared with
npc+/+ neurons, according to Student's
t test.
/
neurons (114.9 ± 10.4, 94.3 ± 6.0, and 100.0 ± 8.4 ng of LDL protein/h/mg
cell protein for npc+/+,
npc+/
, and npc
/
neurons, respectively). The hydrolysis of LDL-derived cholesteryl esters was also unaffected by npc genotype. Hydrolysis was
calculated as radioactivity in [3H]cholesterol as a
percentage of total radioactivity in cholesterol plus cholesteryl
esters (79.9 ± 5.1, 82.6 ± 5.1, and 89.3 ± 4.7% for
npc+/+, npc+/
, and
npc
/
neurons, respectively). Thus, neither
uptake nor hydrolysis of LDL-derived cholesteryl esters depended on
npc genotype.
CD). ApoE-free HDLs containing
[3H]cholesteryl oleate or M
CD containing
[3H]cholesterol were incubated with cell bodies of
npc+/+, npc+/
, and
npc
/
neurons. As shown in Fig. 3, the
percentage of HDL-derived cholesterol transported from cell bodies to
distal axons was independent of NPC1. Similarly, the anterograde
transport of cholesterol delivered to the plasma membrane via M
CD
was the same in npc
/
,
npc+/
, and
npc+/+ neurons (Fig. 3). We have shown
previously that in sympathetic neurons cholesterol synthesis is
restricted to cell bodies/proximal axons (28). The rate of cholesterol
biosynthesis, as indicated by the incorporation of
[14C]acetate into cholesterol, was independent of
npc genotype (5.8 ± 1.1, 7.2 ± 1.1, and 6.2 ± 0.5 dpm/h/mg protein, for npc+/+,
npc+/
, and npc
/
neurons, respectively). The amount of endogenously synthesized cholesterol (labeled from [14C]acetate) transported into
distal axons was, however, reduced by ~18% (p < 0.025) in npc
/
neurons compared with
npc+/+ neurons (Fig. 3).
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Fig. 4.
Anterograde transport of cholesterol in
neurons is inhibited by U18666A but not by progesterone. Cell
bodies/proximal axons of sympathetic neurons in compartmented dishes
were incubated for 5 h with 1.5 µM U18666A or 20 µM progesterone. Cell bodies/proximal axons were then
incubated for 44 h with 10 mCi/ml [14C]acetate (to
label endogenously synthesized cholesterol) or LDLs containing
[3H]cholesteryl oleate (0.1 mg protein/ml) or HDLs
containing [3H]cholesteryl oleate (0.19 mg protein/ml) in
medium supplemented with delipidated rat serum in the absence
(filled bars) or the presence of 1.5 µM
U18666A (shaded bars) or 20 µM progesterone
(open bars). Cellular material was harvested separately from
cell body- and distal axon-containing compartments. Lipids were
extracted and separated by thin layer chromatography, and radioactivity
was measured in unesterified cholesterol. The data are the means ± S.E. of three independent experiments and are expressed as
radioactivity in unesterified cholesterol in distal axons as
percentages of radiolabeled cholesterol in cell bodies/proximal axons + distal axons. *, p < 0.002 compared with cells treated
with neither progesterone nor U18666A.
/
Neurons When Cholesterol Biosynthesis Is Inhibited--
Under normal
culture conditions sympathetic neurons do not require an exogenous
source of cholesterol for axonal growth (32). Moreover, the rate of
axonal regeneration following axotomy (removal of distal axons) is the
same when the neurons are cultured in medium containing full serum
(0.269 ± 0.033 mm/day) or lipoprotein-deficient serum (0.296 ± 0.031 mm/day). Our laboratory previously showed that inhibition of
cholesterol biosynthesis in rat sympathetic neurons significantly
reduces the rate of axonal extension (32) but that normal growth is
restored when LDLs are added to either cell bodies or distal axons (32,
66). Thus, when the supply of cholesterol from endogenous synthesis is
insufficient to sustain normal axonal growth, cholesterol can be used
from exogenously supplied lipoproteins. This "rescue" mechanism of
LDLs presumably depends on trafficking of endocytosed LDL cholesterol,
which we predicted would be perturbed in
npc
/
neurons. We therefore determined
whether or not LDLs restored normal axonal growth to
npc
/
neurons in which cholesterol synthesis
was inhibited by pravastatin. As previously observed in rat sympathetic
neurons (32), pravastatin inhibited axonal elongation of
npc+/+ (Fig.
5A),
npc+/
(Fig. 5B), and
npc
/
(Fig. 5C) murine sympathetic
neurons. After 6 days, the inhibition of axonal extension by
pravastatin was somewhat less pronounced in
npc
/
neurons (29.1 ± 3.2%) than in
npc+/+ and npc+/
neurons (39.7 ± 3.1 and 43.4 ± 2.4%, respectively. LDLs
(0.1 mg protein/ml) restored normal axonal growth to
pravastatin-treated npc+/+ and
npc+/
neurons (Fig. 5, A and
B, respectively) but not to pravastatin-treated npc
/
neurons (Fig. 5C). The
addition of LDLs to neurons of all three npc genotypes, in
the absence of pravastatin, did not alter the rate of axonal extension
(not shown). Fig. 5D summarizes these data and shows that
npc
/
neurons have a defect in the metabolism
of LDL-derived cholesterol that becomes apparent when the neurons rely
on exogenously supplied cholesterol for growth.
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Fig. 5.
Neither LDLs nor HDLs support axonal
extension of pravastatin-treated
npc /
neurons. Cell
body-containing compartments of 12-day-old sympathetic neurons were
incubated overnight in medium containing delipidated serum ± 50 µM pravastatin. Distal axons were then removed, and cell
bodies were given medium containing delipidated serum (open
circles) or the same medium containing 50 µM
pravastatin (filled diamonds) or 50 µM
pravastatin with 0.1 mg LDL protein/ml (filled triangles).
The axons were allowed to regenerate, and axonal extension was
measured. Fresh media were supplied to the center compartment every 3 days. A, npc+/+. B,
npc+/
. C, npc
/
. The data are
the means ± S.E. of four independent experiments in each of which
30 tracks were measured per dish for two cultures of each genotype and
each treatment. D, restoration of growth of
pravastatin-treated neurons after 6 days in the presence of LDLs or
HDLs. The values for the restoration of growth are given as the
differences between axonal extension of neurons incubated with
pravastatin, with or without lipoproteins, as percentages of the
difference between axonal extension of neurons incubated with or
without pravastatin in the absence of lipoproteins. The data for LDLs
are from the experiments in A-C and are the means ± S.E. of four independent experiments. The values for HDLS are from a
parallel experiment performed in triplicate dishes with 30 tracks
measured per dish. *, p < 0.0001 compared with
npc+/+ neurons.
/
neurons. However, although HDL
cholesterol (from apoE-free HDLs containing the same amount of
cholesterol as the LDLs used in Fig. 5) was transported from cell
bodies to distal axons (Fig. 3), this source of cholesterol did not
restore normal axonal extension to pravastatin-treated neurons of any
of the three npc genotypes (Fig. 5D).
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Fig. 6.
Axonal extension of neurons incubated with
U18666A or progesterone. Wild-type neurons were axotomized then
allowed to regenerate for 6 days. A, cell bodies were given
medium containing delipidated rat serum (closed bar) or the
same medium containing 1.5 µM U18666A (open
bar) or 1.5 µM U18666A + 0.1 mg of LDL protein/ml
(hatched bar). B, medium containing delipidated
rat serum with 20 µM progesterone (closed bar)
or the same medium containing 50 µM pravastatin
(open bar) or 50 µM pravastatin + 0.1 mg of
LDL protein/ml (hatched bar). C, cell bodies of
rat sympathetic neurons were given medium containing delipidated rat
serum or the same medium containing 50 µM pravastatin and
the indicated concentrations of LDLs. Axonal extension was measured on
day 3 after axotomy. All of the data are the means ± S.E. of one
experiment performed in triplicate dishes with 30 tracks measured/dish.
A total of three independent experiments were performed with similar
results. *, p < 0.05; **, p < 0.005;
***, p < 0.0005.
Phenotypes elicited in sympathetic neurons by progesterone U18666A, and
a lack of functional NPC1
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
neurons, whereas the transport of
endogenously synthesized cholesterol is modestly impaired; (iii)
despite the apparently normal anterograde transport of LDL cholesterol,
this source of cholesterol is not available for growth of
npc
/
neurons; and (iv) neither U18666A nor
progesterone phenocopies NPC1 deficiency in neurons.
CD-complexed cholesterol into distal axons was not
significantly different among npc+/+,
npc+/
, and npc
/
sympathetic neurons. However, we observed a small (~18%) but statistically significant decrease in the transport of endogenously synthesized cholesterol into distal axons of
npc
/
neurons. The apparently normal
anterograde transport of LDL-derived cholesterol in
npc
/
neurons was unexpected because
lysosomes and late endosomes (the sites of accumulation of LDL
cholesterol in npc
/
fibroblasts) are
commonly thought to be restricted to neuronal cell bodies (68).
/
neuronal cell bodies, and no
LDL-derived cholesteryl esters are found in distal axons of either
npc genotype. In addition, in sympathetic
neurons from LDL receptor null mice hydrolysis and anterograde
transport of LDL-derived cholesteryl esters were reduced by 86%
compared with wild-type neurons. Thus, the majority of LDL-derived
cholesterol taken up by cell bodies is processed via the endocytic
pathway. We therefore hypothesize that in
npc
/
neurons LDL-derived cholesteryl esters
reach late endosomes (that would normally contain NPC1) in cell bodies
and that these endosomes are delivered to axons independent of NPC1.
This hypothesis is supported by our observation that NPC1 and LAMPI are
present in distal axons.
/
neurons. However, the initial delivery
of cholesterol to the plasma membrane, according to the model of Cruz
et al., would occur in a rapid step that would not have been
specifically monitored by the long term transport assay we used. In
NPC1-deficient fibroblasts, late endosomes have been
proposed to fuse with lysosomes and be transformed into multilamellar
hybrid vesicles (9). We hypothesize that in
npc
/
neurons, similar cholesterol-laden late
endosomes are formed in cell bodies and move into axons. However,
egress of cholesterol from these endosomes might be impaired, which
would result in an inability of LDL-derived cholesterol to reach the
location required for supporting growth. Hollenbeck and co-workers have indicated that at least part of the endosomal pathway is present in
axons (70, 71), although others have concluded that lysosomes and late
endosomes, as well as their associated degradative enzymes, are
restricted to neuronal cell bodies (62, 68). It is possible that the
movement of at least a subset of NPC1-containing late endosomes into
axons is a normal mechanism for delivery of cholesterol, in accordance
with our observation that NPC1 and LAMP1 are present in axons.
/
neurons. We found that LDLs restored a
normal rate of axonal extension to pravastatin-treated
npc+/+ and npc+/
neurons but not npc
/
neurons. Therefore,
although a normal amount of LDL cholesterol arrives in distal axons
from cell bodies of npc
/
neurons, this
source of cholesterol cannot be used for axonal extension. In line with
these observations, Goodrum and Pentchev (38) have demonstrated that
peripheral nerve regeneration, which is thought to involve the
endocytic uptake of lipoproteins, is impaired in
npc
/
mice.
/
neurons (summarized in Table I). For
example, U18666A, but not NPC1 deficiency or progesterone, strongly
inhibits the anterograde transport of cholesterol derived from
lipoproteins. In addition, U18666A inhibits axonal growth, whereas
progesterone and NPC1 deficiency do not. Moreover, U18666A, but not
progesterone or NPC1 deficiency, inhibits cholesterol synthesis to an
extent similar to pravastatin.
/
neurons
and in wild-type neurons exposed to U18666A, LDL cholesterol is not
delivered to the site required for axonal growth.
/
neurons but
cannot be used effectively for axonal growth, presumably because the
cholesterol is not released from endosomes. In addition, transport of
endogenously synthesized cholesterol into axons of npc
/
neurons is impaired. These findings
support our previous observations that the amount of cholesterol in
cell bodies of npc
/
neurons is increased
compared with that in npc+/+ neurons, whereas
the cholesterol content of distal axons is decreased (49). This deficit
in axonal cholesterol might become more pronounced over the long life
of neurons in intact animals, particularly if the majority of axonal
cholesterol originated from endogenous synthesis, as appears to be the
case in cultured sympathetic neurons (32, 66) and the brain (33-35).
Eventually, a reduction in the cholesterol content of NPC1-deficient
axons in vivo, rather than an accumulation of cholesterol in
cell bodies, might result in defective axonal regeneration and/or
remodeling, which might account, at least in part, for the progressive
neurodegeneration in NPC patients.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susanne Lingrell and Russ Watts for excellent technical assistance and Dr. G. Francis (University of Alberta) for providing human lipoproteins.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Canadian Institutes for Health Research and the Ara Parseghian Medical Research Foundation.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.
¶ Supported by postdoctoral fellowships from the Alberta Heritage Foundation for Medical Research and the Deutsche Forschungsgemeinschaft (Forschungsstipendium KA1578/1).
** Medical Scientist of the Alberta Heritage Foundation for Medical Research.
Holder of the Canada Research Chair in Molecular and Cell
Biology of Lipids.
¶¶ To whom correspondence should be addressed: 332 Heritage Medical Research Centre, University of Alberta, Edmonton, AB T6G 2S2, Canada. Tel.: 780-492-7250; Fax: 780-492-3383; E-mail: jean.vance@ualberta.ca.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M205406200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NPC, Niemann Pick C;
HDL, high density lipoprotein;
LAMP1, lysosome-associated membrane
protein 1;
LDL, low density lipoprotein;
MCD, methyl
-cyclodextrin;
NGF, nerve growth factor;
PBS, phosphate-buffered
saline;
U18666A, 3-
-[2-diethylaminoethoxy]androst-5-ene-17-one.
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