Journal of Neurochemistry, 2002, 83, 1154–1163
Cholesterol accumulates in cell bodies, but is decreased in distal
axons, of Niemann–Pick C1-deficient neurons
Barbara Karten,*, ,1
Dennis E. Vance,*,à,2,3 Robert B. Campenot§,2 and Jean E. Vance*,
*Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids and Departments of Medicine,
àBiochemistry and §Cell Biology, University of Alberta, Edmonton, Alberta, Canada
Abstract
Niemann–Pick type-C (NPC) disease is characterized by a
progressive loss of neurons and an accumulation of uneste-
rified cholesterol within the endocytic pathway. Unlike other
tissues, however, NPC1-deficient brains do not accumulate
cholesterol but whether or not NPC1-deficient neurons accu-
mulate cholesterol is not clear. Therefore, as most studies on
cholesterol homeostasis in NPC1-deficient cells have been
performed in fibroblasts we have investigated cholesterol
homeostasis in cultured murine sympathetic neurons lacking
functional NPC1. These neurons did not display obvious
abnormalities in growth or morphology and appeared to
respond normally to nerve growth factor. Filipin staining
revealed numerous cholesterol-filled endosomes/lysosomes
in NPC1-deficient neurons and the mass of cholesterol in cell
bodies was greater than in wild-type neurons. Surprisingly,
Niemann–Pick type-C (NPC1) disease is a fatal, autosomal
recessive disorder that leads to premature death (Pentchev
et al. 1995; Vanier and Suzuki 1998). The disease is caused
in 95% of cases by a mutation in the NPC1 gene resulting in
a lack of function of the NPC1 protein (Carstea et al. 1997).
The remaining 5% of NPC patients have a defect in the HE1
gene (Naureckiene et al. 2000). Patients with NPC disease
exhibit increasing loss of motor control, seizures and other
neuropathological symptoms (Pentchev et al. 1995; Vanier
1999). Similar to lysosomal storage diseases, NPC disease is
associated with axonal abnormalities (spheroids, meganeur-
ites and axonal dystrophy), demyelination, and formation of
neurofibrillary tangles. With the exception of neurofibrillary
tangle formation, similar changes are observed in a murine
model for NPC disease (Higashi et al. 1993; German et al.
2001).
The exact function of the NPC1 protein is still unknown.
Essentially all biochemical studies on NPC1-deficient cells
have been performed in non-neuronal cells such as fibro-
blasts and mutant CHO cells (Cadigan et al. 1990; Liscum
1154 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1154–1163
however, the cholesterol content of NPC1-deficient and
wild-type neurons as a whole was the same. This apparent
paradox was resolved when the cholesterol content of NPC1-
deficient distal axons was found to be less than of wild-type
axons. Cholesterol sequestration in cell bodies did not depend
on exogenously supplied cholesterol since the cholesterol
accumulated before birth and did not disperse when neurons
were cultured without exogenous cholesterol. The altered
cholesterol distribution between cell bodies and axons
suggests that transport of cholesterol, particularly that
synthesized endogenously, from cell bodies to distal axons is
impaired in NPC1-deficient neurons.
Keywords: axons, cholesterol, filipin, neurons, Niemann–
Pick C, U18666A.
J. Neurochem. (2002) 83, 1154–1163.
et al. 1989) in which unesterified cholesterol accumulates in
late endosomes/lysosomes. In these cells, the transport of low
density lipoprotein (LDL)-derived cholesterol from late
endosomes/lysosomes to the plasma membrane is defective;
Received July 5, 2002; revised manuscript received August 29, 2002;
accepted September 6, 2002.
Address correspondence and reprint requests to Dr Jean E. Vance, 332
Heritage Medical Research Center, University of Alberta, Edmonton
Alberta T6G 2S2, Canada. E-mail: jean.vance@ualberta.ca
1
Supported by postdoctoral fellowships from the Alberta Heritage
Foundation for Medical Research and the Deutsche Forschungsge-
meinschaft (Forschungsstipendium KA1578/1).
2
Medical Scientists of the Alberta Heritage Foundation for Medical
Research.
3
Holder of the Canada Research Chair in Molecular and Cell Biology of
Lipids.
Abbreviations used: HDL, high density lipoprotein; LAMP1, lyso-
some-associated membrane protein 1; LDL, low-density lipoprotein;
NGF, nerve growth factor; NPC, Niemann–Pick type C; PBS, phosphate-
buffered saline; SDS, sodium dodecyl sulfate; U18666A, 3-b-(2-diethyl-
aminoethoxy)androst-5-ene-17-one.

it now also appears that the trafficking of endogenously
synthesized cholesterol is impaired (Lange and Steck 1998;
Cruz and Chang 2000; Lange et al. 2000).
In the brain, the majority of cholesterol is derived from
endogenous synthesis since plasma lipoproteins do not
cross the blood–brain barrier (Danik et al. 1999; Turley
et al. 1996; Turley et al. 1998). However, the increased
need for cholesterol during nerve regeneration is believed to
be met by receptor-mediated uptake of apo E-containing
lipoproteins secreted by endoneurial macrophages which
salvage cholesterol released by degenerating neurons and
myelin (Goodrum et al. 2000). In NPC1-deficient mice, this
recycling mechanism for cholesterol has been reported to be
impaired (Goodrum and Pentchev 1997; German et al.
2002). In most tissues of NPC patients, cholesterol
accumulates but the relationship between this accumulation
and the neurological manifestations is not understood. In
addition to cholesterol, glycosphingolipids also accumulate
in lysosomes/late endosomes of NPC1-deficient cells (Tan-
iguchi et al. 2001; Zervas et al. 2001) and the concentration
of glycosphingolipids in NPC brains is increased (Vanier
1999). In contrast, in the brains of NPC patients the
cholesterol content decreases with age (Vanier 1999; Xie
et al. 1999). Thus, it has been suggested that NPC1-
deficient neurons do not accumulate cholesterol (Elleder
et al. 1985). However, Dietschy and coworkers have
proposed that the decrease in cholesterol in NPC1-deficient
brains is the result of an extensive loss of myelin which is
rich in cholesterol (Xie et al. 2000), and that cholesterol
does accumulate in neurons and glial cells. On the other
hand, Henderson et al. (2000) have found that the choles-
terol content of cultured embryonic striatal neurons from
NPC1-deficient mice is normal.
In light of the conflicting and incomplete data on whether
or not cholesterol accumulates in neurons lacking functional
NPC1, and since NPC disease is primarily a neurological
disorder, we have examined axonal growth and cholesterol
homeostasis in primary neurons from NPC1-deficient mice.
We show that NPC1 deficiency results in an accumulation of
cholesterol in cell bodies but a reduction in the cholesterol
content of distal axons suggesting that the transport of
cholesterol from cell bodies into distal axons is impaired in
NPC1-deficient neurons.
Materials and methods
Materials
Cell culture media and B27 supplement were purchased from
Invitrogen (Burlington, ON, Canada). Other cell culture materials
were from BD Biosciences (Bedford, MA, USA). The rabbit anti-
human polyclonal NPC1 antibody used for immunoblotting was a
generous gift from Dr D. Ory (Washington University, St. Louis,
MO, USA) and was raised against a peptide consisting of amino
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Cholesterol accumulation in npc–/– neurons 1155
acids 1261–1272 of human NPC1. A rabbit anti-mouse polyclonal
antibody raised against amino acids 1254–1273 of murine NPC1
was provided by Dr W. S. Garver (University of Arizona, Tucson,
AZ, USA) and was used for the immunocytochemical studies. The
rat-anti mouse LAMP1 antibody was from PharMingen (Lexing-
ton, KY). Alexa Fluor 488-labeled goat anti-rabbit IgG and Texas
Red-labeled goat anti-rat IgG were from Molecular Probes
(Eugene, OR, USA). U18666A [3-b-(2-diethylaminoethoxy)andr-
ost-5-en-17-one] was purchased from Biomol Research Laborator-
ies (Plymouth Meeting, PA, USA). Other materials for gel
electrophoresis and immunoblotting were from Bio-Rad (Mississ-
auga, ON, Canada). Monoclonal antib-tubulin antibody (T 4026),
filipin complex, and cholesterol were purchased from Sigma (St.
Louis, MO, USA). Mouse 2.5S nerve growth factor was from
Alomone Laboratories Ltd. (Jerusalem, Israel). Rat serum was
provided by the University of Alberta Animal Services. Delipi-
dated rat serum was prepared by the method of Cham and
Knowles (Cham and Knowles 1976). All other materials were
from Sigma, unless stated otherwise.
Primary cultures of sympathetic neurons
from NPC1-deficient mice
A breeding colony of heterozygous Balb/cNctr-npcN/+mice, ori-
ginally from the Jackson Laboratories (Bar Harbor, ME, USA), was
established at the University of Alberta. Mice were maintained
under temperature-controlled conditions with a 12-h light : 12-h
dark cycle. Hereafter, mice homozygous or heterozygous for the
mutation in the npc1 gene will be referred to as npc–/– or npc+/–,
respectively, whereas wild-type mice will be designated as npc+/+.
The genotype was determined from genomic DNA extracted from
tail clippings of 1-day-old mouse pups in a PCR reaction as
described (Loftus et al. 1997). All experiments were performed by
comparing littermates of the three genotypes and were approved by
the Health Sciences Animal Welfare Committee of the University of
Alberta. Superior cervical ganglia were dissected from one-day-old-
mouse pups. Ganglia of each mouse pup were placed separately into
sterile microfuge tubes containing L15 medium with 10% rat serum
and 50 ng/mL NGF and maintained at 4°C overnight, but for no
longer than 24 h, prior to dissociation. This time period allowed
genotyping of tail clippings from each mouse pup to be performed
and consequently ganglia of one genotype could be pooled prior to
dissociation. Neurons 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 (Karten et al. 2002).
Compartmented cultures were assembled as described previously
(Campenot 1979; Campenot 1992). Briefly, a Teflon divider (Tyler
Research Instruments, Edmonton, AB, Canada) was sealed with
silicon grease to a 35-mm collagen-coated dish thereby partitioning
the dish into three compartments. Dissociated neurons were plated
in the center compartment. Within 2–3 days axons elongated along
the tracks, crossing under silicon grease barriers into left- and right-
side compartments. Thus, the center compartment contained cell
bodies and proximal axons whereas side compartments contained
pure distal axons. No exchange of fluid occurs between compart-
ments (Campenot 1979). L15 medium with penicillin/streptomycin,
L-glutamine, glucose and the additives prescribed by Hawrot and
Patterson (1979), including bicarbonate and methylcellulose, was
used as basal medium to which was added 2.5% rat serum for the

1156 B. Karten et al.
center compartment and 100 ng/mL NGF for the side compart-
ments. During the first week after plating, 10 lM cytosine
arabinoside and 20 ng/mL NGF were present in the center
compartment. Neurons were cultured for 10–12 days prior to the
start of all experiments. In some experiments, neurons were placed
in serum-free medium consisting of a 1 : 1 mixture of Dulbecco’s
modified Eagle’s medium and Ham’s F10 medium containing 0.4%
methylcellulose (w/v) with the same antibiotics and additives except
that a B27 supplement mixture was used instead of serum.
Measurement of axonal extension
Axonal extension was measured in compartmented dishes after
removal of distal axons (a process hereafter termed ÔaxotomyÕ) from
the side compartments with a jet of sterile distilled water (Campenot
1992). The wash was repeated twice to remove all traces of distal
axons from the side compartments without disturbing the collagen.
Axonal extension was measured using a Nikon Diaphot inverted
microscope equipped with a MD2 microscope digitizer (Accustage
Corp., Minneapolis, MN, USA) which tracks stage movements to an
accuracy of ± 0.005 mm. In each culture, 16 tracks were measured
in each side compartment. As an independent indicator of axonal
growth, the amount of axonal b-tubulin in distal axons was assessed
by immunoblotting. Proteins from distal axons were resolved on
10% polyacrylamide gels containing 0.1% SDS under reducing
conditions, then transferred to a nitrocellulose membrane and
probed with anti-b-tubulin antibody (dilution 1 : 1000). Immuno-
blots were developed using ECL reagent (SuperSignal West Dura
Extended, Pierce, Rockford, IL, USA).
Immunoblotting of NPC1 protein
Neurons were harvested into ice-cold homogenization buffer consist-
ing of 20 mM Tris-HCl, 1 mM EDTA, 0.25 mM sucrose (pH 7.4) with
1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail
(Complete Mini, Roche Diagnostics, Mannheim, Germany). Samples
were homogenized in a glass homogenizer and nuclei and unbroken
cells were removed from the homogenate by centrifugation at 1500 g
for 10 min at 4°C. The supernatant was centrifuged in a TLA100.2
rotor (Beckman Instruments, Mississauga, ON, Canada) 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. Proteins were resolved on 6% polyacrylamide gels
containing 0.1% sodium dodecyl sulfate (SDS) then transferred to
nitrocellulose and immunoblotted using anti-human NPC1 antibody
(dilution 1 : 1000) and peroxidase-conjugated goat anti-rabbit IgG
(dilution 1 : 10 000) as secondary antibody. Immunoreactivity was
detected with ECL reagent (ECLWestern Blotting System, Amersham
Biosciences, Piscataway, NJ, USA).
NGF deprivation and neuronal survival
Neurons in 96-well plates were washed three times with L15
medium containing 0.4% methylcellulose with a 10-min incubation
at 37°C between washes to ensure diffusion of the unstirred water
layer above the cells. Remaining traces of NGF were removed by
incubation for 30 min with 100 lL medium containing 12 nM anti-
NGF antibody (Cedarlane Laboratories Ltd, Hornby, ON, Canada).
The cells were washed, then medium containing the indicated
concentrations of NGF was added for 40 h. Neuronal survival
was assessed based on the ability of the neurons to convert
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3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
(MTT tetrazolium salt) into an insoluble formazan product using
the CellTiter 96Ò Non-Radioactive Cell Proliferation Assay
(Promega, Madison, WI, USA).
Immunocytochemistry and filipin staining
Neurons in compartmented dishes or on collagen-coated coverslips
were fixed for 20 min at room temperature in 4% paraformaldehyde,
then blocked in 10% goat serum in phosphate-buffered saline
(PBS) ± 50 lg/mL filipin for 2 h at room temperature. For
immunocytochemical localization of LAMP1 and NPC1 the primary
antibodies used 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 351 nm (filipin), 488 nm (Alexa Fluor
488), and 543 nm (Texas Red).
Cholesterol biosynthesis
Neurons were cultured in 48-well plates for 12 days then incubated
for 24 h with 0.4 lCi/mL [14C]acetate in basal medium containing
2.5% rat serum and 100 ng/mL NGF. Lipids were extracted with
hexane/isopropanol 3 : 2 (v/v) and cholesterol was separated by
thin-layer chromatography in the solvent system hexane/diisopropyl
ether/acetic acid 65 : 35 : 4 (v/v/v).
Determination of cellular cholesterol content
Cellular lipids were extracted twice with 2.5 mL hexane/isopropanol
3 : 2 (v/v) after which an internal standard, 5a-cholestane, was
added. Unesterified cholesterol was converted to its trimethylsilyl
ether derivative by reaction with bis(trimethylsilyl)trifluoroaceta-
mide containing 1% trimethylchlorosilane in acetone for 15 min at
50°C. The trimethylsilyl derivative was analyzed by gas–liquid
chromatography.
Other methods
Human lipoproteins were isolated by ultracentrifugation of plasma
of normolipidemic volunteers in a KBr gradient (Sattler et al. 1994).
Concentrations of lipoproteins are given as mg protein/mL. Cellular
protein was determined using the BCA Protein Assay (Pierce,
Rockford, IL, USA).
Results
Sympathetic neurons express NPC1 protein
Even though npc–/– mice have no apparent phenotype at the
time of birth, only 12% of offspring generated by crossing
npc+/– mice were homozygous for the npc1 mutation,
indicating a prenatal lethality caused by the npc1 mutation.
This embryonic lethality apparently occurs after embryonic
day 16 since Henderson and coworkers have reported that the
expected 25% of pups taken at this time are of the npc–/–
genotype (Henderson et al. 2000).

Fig. 1 NPC1 is expressed in sympathetic neurons. Membrane-
enriched fractions were prepared from rat sympathetic neurons
maintained for 12 days in 24-well plates (lane 1: SCG), and from
homogenates of brain (lanes 2–4) and liver (lanes 5–7) from 5-week-
old npc+/+, npc+/– and npc–/– mice. Proteins were resolved by
electrophoresis on polyacrylamide gels and immunoblotted with anti-
human NPC1 antibody. Ten micrograms of protein were applied to
each lane.
NPC1 mRNA has been detected in several types of
neurons in the brain using in situ hybridization techniques
(Prasad et al. 2000) but, to our knowledge, the expression
of NPC1 protein in neurons has not been reported. We
therefore confirmed that sympathetic neurons express NPC1
protein by immunoblotting using a polyclonal anti-human
NPC1 antibody. As shown in Fig. 1, lane 1, an immuno-
reactive protein of
170 kDa, presumed to be NPC1,
comigrated with a protein from brains and livers of wild-
type mice (Fig. 1, lanes 2 and 5). This protein was less
abundant in brain and liver homogenates from npc+/– mice
(Fig. 1, lanes 3 and 6), and was not detected in npc–/– mice
(Fig. 1, lanes 4 and 7).
Neurons from npc–/– mice exhibit normal morphology
and growth
To investigate if axonal growth of neurons is defective in
the absence of NPC1, we established conditions for culture
of primary sympathetic neurons from the superior cervical
ganglia of one-day-old npc–/–, npc+/– and npc+/+ mice in
three-compartment dishes (Karten et al. 2002) by modifying
techniques previously used for rat sympathetic neurons
(Campenot 1979). In this culture system, cell bodies and
distal axons are maintained in separate fluid environments.
Thus, cell bodies/proximal axons and distal axons can be
treated and harvested separately, and axonal extension can
be measured accurately (Campenot 1992). When viewed
under the light microscope, sympathetic neurons of the
three npc genotypes were indistinguishable: yields of
neurons per ganglion were comparable, npc–/– neurons
grew well in compartmented dishes and the number of
axons appeared to be the same. When distal axons were
removed and allowed to regenerate for 6 days, no differ-
ences in axonal extension were observed among npc–/–,
npc+/– and npc+/+ neurons (Fig. 2a). As confirmation that
axonal growth was the same in neurons of the 3 npc
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Cholesterol accumulation in npc–/– neurons 1157
Fig. 2 Axonal extension is not dependent on NPC1 protein. Murine
sympathetic neurons were axotomized then axons were allowed to
regenerate in medium containing delipidated serum. (a) Axonal
extension. s, npc+/+; h, npc+/–; n, npc–/–. Data represent means ± SE
of four independent experiments with duplicate dishes and 30 tracks
measured per dish. (b) On day 7 after axotomy, distal axons were
harvested and proteins analyzed by polyacrylamide gel electro-
phoresis and immunoblotting with antib-tubulin antibody. Each lane
contains protein from distal axons of 1 dish of neurons of each
genotype. One representative experiment of four similar experiments
is displayed.
genotypes we demonstrated by immunoblotting that the
amount of b-tubulin, the most abundant cytoskeletal protein
of axons, was the same in distal axons of npc+/+, npc+/–
and npc–/– neurons (Fig. 2b).
Survival of npc–/– neurons is not impaired
in low concentrations of NGF
Sympathetic neurons depend on NGF for survival and
growth (reviewed in (Sofroniew et al. 2001)). Thus, our
observation that npc–/– neurons grew normally in compart-
mented cultures indicated that retrograde signaling of NGF
was not severely impaired. To test whether a possible defect
in NGF-dependent signal transduction in npc–/– neurons was
masked by the high concentrations of NGF (100 ng/mL)
routinely used, we assessed neuronal survival in low
concentrations of NGF, and also after deprivation of NGF
for 40 h (Fig. 3). Although fewer neurons survived when
the NGF concentration was 0.5 ng/mL compared to 5 or
50 ng/mL, the percentage of neurons of the three npc
genotypes that survived at each NGF concentration was the
same. Thus, the sensitivity of sympathetic neuronal survival
to deprivation of NGF is not dependent on NPC1.

1158 B. Karten et al.
Fig. 3 Dependence of survival of npc+/+, npc+/– and npc–/– neurons on
NGF concentration. Five-day-old neurons in 96-well plates were
washed thoroughly and residual NGF was removed by incubation with
12 nM anti-NGF antibody. The neurons were then incubated for 40 h in
medium containing serum and the indicated concentrations of NGF.
The MTT survival assay was used to distinguish live and dead cells
(stained versus not stained, respectively). Data are expressed as
percentage of live cells per total cells counted and are means ± SE of
an experiment performed in quadruplicate. Filled bars: npc+/+, striped
bars: npc+/–, open bars: npc–/–.
Cholesterol-laden vesicles accumulate in cell bodies
of npc–/– neurons
We next compared the amount of unesterified cholesterol in
npc–/– and npc+/+ neurons by directly measuring cholesterol
mass and by performing confocal microscopy on neurons
stained with filipin, a dye that forms a fluorescent complex
npc +/+ npc +/-
Filipin
LAMP1
NPC1
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1154–1163
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with unesterified cholesterol. Figure 4 shows that filipin
fluorescence of 12-day-old npc+/+ neurons was mainly
restricted to the plasma membrane. In contrast, a bright
punctate intracellular staining pattern, indicative of cytoplas-
mic cholesterol-laden vesicles, was evident in npc–/– neurons.
The filipin staining pattern of npc+/– neurons (Fig. 4) was
intermediate between that of npc+/+ and npc–/– neurons.
Filipin staining of neurons incubated with LDLs or high
density lipoproteins (HDLs) revealed similar fluorescence
patterns (data not shown).
In fibroblasts (Roff et al. 1992) and CHO cells (Cadigan
et al. 1990) lacking functional NPC1, the cholesterol-laden
vesicles also contain the lysosomal membrane-associated
protein 1 (LAMP1) (Chen et al. 1985). The punctate
distribution of LAMP1 was similar in neurons of all three
npc genotypes (Fig. 4). There was a pronounced colocaliza-
tion of filipin fluorescence and LAMP1 immunoreactivity in
npc–/– neurons that was not evident in npc+/+ neurons
(Fig. 4). We included an anti-murine NPC1 antibody in our
immunostaining protocol to confirm that NPC1 was
expressed in npc+/+ and npc+/– neurons, but not in npc–/–
neurons (Fig. 4). These data indicate that cholesterol accu-
mulates in late endosomes/lysosomes of npc–/– sympathetic
neurons, as has been shown in NPC1-deficient fibroblasts.
Progesterone and the amphiphilic amine U18666A have
been reported to induce an NPC-like phenotype (i.e.
npc -/-
Fig. 4 Cholesterol-filled vesicles accumu-
late in cell bodies of npc–/– neurons. Sym-
pathetic neurons from npc+/+ (left-hand
panels), npc+/– (middle panels), and npc–/–
(right-hand panels) mice were stained with
filipin and incubated with antibodies raised
against LAMP1 or NPC1, then examined by
confocal microscopy. Scale bar ¼ 20 lm.
The experiment was performed four times
with similar results.
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Cholesterol accumulation in npc–/– neurons 1159

Fig. 5 Cholesterol-filled vesicles accumu-

late in cell bodies of neurons treated with
U18666A or progesterone in the presence
of LDLs. Wild-type murine neurons were
incubated for 2 days in medium containing
delipidated serum (a), or the same medium
supplemented with 20 lM progesterone and
0.1 mg/mL LDLs (b), 1.5 lM U18666A and
0.1 mg/mL LDLs (c), 20 lM progesterone
(d), or 1.5 lM U18666A (e). Filipin staining
was examined by confocal microscopy.
Scale bar ¼ 20 lm. The experiment was
performed three times with similar results.

accumulation of LDL-derived cholesterol in intracellular terol in cell bodies/proximal axons was compensated by a
vesicles) in fibroblasts (Liscum and Faust 1989; Butler et al. reduced amount of cholesterol in distal axons. We therefore
1992). To determine if these drugs also caused an accumu- measured the cholesterol content of cell bodies/proximal
lation of cholesterol in neurons we incubated wild-type
neurons for 2 days with LDLs in the absence or presence of
progesterone or U18666A. In the absence of these two drugs,
the plasma membrane stained intensely with filipin (Fig. 5a).
In contrast, incubation of the neurons with progesterone
(Fig. 5b) or U18666A (Fig. 5c) induced an intracellular
punctate filipin staining pattern that mainly colocalized with
LAMP1 (not shown) and that was similar to that seen in
npc–/– neurons (Fig. 4). Even when the neurons were
cultured in delipidated serum, U18666A or progesterone
caused accumulation of cholesterol-laden vesicles (Figs 5d
and e, respectively).

Cholesterol content of npc–/– neurons

To investigate if the increased filipin fluorescence we
observed in npc–/– neurons (Fig. 4) reflected an increase in
cholesterol content, we measured cholesterol mass in neurons
of the three npc genotypes grown in 48-well plates for
12 days. Surprisingly, the mass of cholesterol (lg choles-
terol/mg protein) in npc–/– neurons was not statistically
significantly different from that in npc+/+ neurons (Fig. 6a).
To address the possibility that the high intensity of filipin
staining was due autofluorescence of lipid-filled vesicles, we
performed confocal microscopy on npc–/– neurons in the Fig. 6 Cholesterol content and distribution in sympathetic neurons.
absence of filipin. A punctate fluorescence pattern was Sympathetic npc+/+, npc+/–, and npc–/– neurons were maintained for
12 days in 48-well plates (a) or compartmented culture dishes (b).
observed in the UV excitation range at 351 nm, similar to,
Lipids were extracted and the cholesterol content measured. In (a)
but of much lower intensity (
5%) than, the filipin staining
values are given as lg cholesterol/mg protein and are means ±
in Fig. 4. Therefore, the autofluorescence exhibited by npc–/–
SE of three independent experiments with between four and six wells
neurons is only a minor contributor to the high fluorescence in each experiment. The cholesterol content of distal axons and cell
intensity observed after filipin staining. bodies/proximal axons (b) is given as percentage of the total choles-
We next considered the possibility that the reason that the terol content of cell bodies/proximal axons plus distal axons. Data
cholesterol content of npc–/– neurons as a whole was not are averages ± SE of five independent experiments. *p < 0.00075 for
increased (Fig. 6a) was because the accumulation of choles- npc–/– versus npc+/+ neurons.

Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1154–1163


1160 B. Karten et al.
Fig. 7 Cholesterol-filled vesicles accumulate in 1-day-old neurons and
in neurons cultured in serum-free medium. Sympathetic neurons were
plated on collagen-coated coverslips in serum-free medium. After
either 1 day (a–c) or 9 days (d–f) the neurons were stained with filipin
axons and distal axons separately in compartmented cultures.
Figure 6(b) shows that cell bodies/proximal axons of npc–/–
neurons contained significantly (p < 0.0007) more cholester-
ol than did npc+/+ and npc+/– neurons, in general agreement
with the filipin staining (Fig. 4). In contrast, the amount of
cholesterol in distal axons of npc–/– neurons was significantly
less than in npc+/+ neurons (p < 0.00075). The amount of
cholesterol/mg protein in cell bodies plus that in distal axons
of these compartmented cultures was not significantly
different in npc+/+, npc+/– and npc–/– neurons (21.3 ± 3.2,
24.1 ± 4.2 and 25.5 ± 5.8 lg/mg protein, respectively), in
accordance with the data (Fig. 6a) from neurons grown in
48-well plates. Moreover axonal growth and the amount of
axonal protein were the same in npc+/+ and npc–/– neurons
(Figs 2a and b). Thus, these data show that there is not
merely an accumulation of cholesterol in cell bodies of npc–/–
neurons but that the amount of cholesterol in axons is
reduced and the distribution of cholesterol between cell
bodies/proximal axons and distal axons is altered.
Origin of the sequestered cholesterol
We next attempted to determine whether the cholesterol that
accumulated in cell bodies of npc–/– neurons was derived
from endogenous synthesis or from exogenously supplied
lipoproteins. Figure 4 shows that cholesterol accumulates in
filipin-positive vesicles in npc–/– neurons that had been
cultured in medium containing serum for 12 days. To deter-
mine whether or not the formation of cholesterol-laden ves-
icles was a consequence of the in vitro cell culture conditions
we stained freshly isolated neurons with filipin. Even 1-day-
old npc–/– sympathetic neurons (Fig. 7c), but not npc+/+ or
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and examined by confocal microscopy. (a) and (d) npc+/+, (b) and (e)
npc+/–, (c) and (f) npc–/–. Scale bar ¼ 20 lm. The experiment was
repeated with similar results.
Fig. 8 The rate of incorporation of [14C]acetate into cholesterol is the
same in npc+/+, npc+/– and npc–/– neurons. Neurons cultured in 48-well
plates were incubated for 24 h with [14C]acetate in medium containing
delipidated rat serum. Lipids were extracted and separated by thin-
layer chromatography. Radioactivity was measured in unesterified
cholesterol. Data are expressed as dpm/h/mg cellular protein and
represent means ± SE of three independent experiments.
npc+/– neurons (Figs 7a and b, respectively), showed a
pronounced punctate filipin fluorescence pattern which
persisted after 9 days in serum-free medium (Figs 7d–f).
Clearly, cholesterol accumulates intracellularly in cell bodies
of npc–/– neurons in vivo during gestation and is not rapidly
mobilized from these vesicles. These data suggest that the
cholesterol accumulating in cell bodies is primarily derived
from endogenous synthesis.
Cholesterol synthesis in sympathetic neurons
One explanation for why cholesterol accumulates in NPC1-
deficient neurons is that the rate of cholesterol synthesis
might have been higher in npc–/– neurons than in npc+/+
neurons (Liscum and Faust 1987; Maziere et al. 1987).

Figure 8 shows, however, that the rate of incorporation of
[14C]acetate into cholesterol was similar in neurons of the
three npc genotypes. Only background levels of radioactivity
were detected in cholesteryl esters. Some (
2.5%) of the
labeled cholesterol was released into the medium, in amounts
that were similar in npc+/+, npc+/– and npc–/– neurons
(8.4 ± 1.3, 9.1 ± 1.2 and 9.4 ± 1.2 dpm/mg protein after
24 h, respectively). Thus, the intracellular accumulation of
cholesterol in cell bodies of npc–/– neurons cannot be
ascribed to either a higher rate of cholesterol synthesis or a
decreased release of cholesterol into the medium.
Discussion
Two important new findings concerning the function of
NPC1 in neurons are reported in this study. First, NGF-
dependent survival and axonal growth of NPC1-deficient
sympathetic neurons are normal. Second, although there is no
overall increase in the cholesterol content of npc–/– neurons,
cholesterol accumulates in cell bodies and is partially
depleted from distal axons. Our data also suggest that the
cholesterol accumulating in cell bodies originates primarily
from endogenous synthesis rather than from exogenously
supplied lipoproteins.
NGF-dependent growth of npc–/– neurons is normal
The similar growth and survival characteristics of npc–/– and
npc+/+ neurons indicate that NGF-dependent survival and
growth are not impaired in npc–/– sympathetic neurons.
These findings are of particular interest in light of recent
studies in which npc–/– embryonic striatal neurons (of central
nervous system origin) exhibited an impaired response to
brain-derived neurotrophic factor (Henderson et al. 2000). In
these neurons, tyrosine phosphorylation of TrkB, the receptor
for this neurotrophin, was reduced and fewer branch points
and shorter axons were formed in npc–/–, compared to
npc+/+, neurons (Henderson et al. 2000). The different
growth characteristics and responsiveness to neurotrophins
between striatal and sympathetic neurons from npc–/– mice
might be because the neurotrophin receptors, TrkA and TrkB,
are activated by different growth factors, NGF and brain-
derived neurotrophic factor, respectively. Since the retro-
grade transport of NGF after binding to the TrkA receptor is
not required for survival of sympathetic neurons (MacInnis
and Campenot 2002), NGF-induced signal transduction
might still occur normally even if NPC1-deficiency affected
retrograde transport processes in general.
Cholesterol accumulation in NPC1-deficient neurons
One of the most obvious abnormalities in npc–/– fibroblasts
and most NPC1-deficient tissues is the accumulation of
unesterified cholesterol in late endosomes/lysosomes (Sokol
et al. 1988; Coxey et al. 1993). In contrast, the cholesterol
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1154–1163
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Cholesterol accumulation in npc–/– neurons 1161
content of NPC1-deficient brains decreases with age (Vanier
1999; Xie et al. 1999). Consequently, it was proposed that
npc–/– neurons do not accumulate cholesterol (Pentchev et al.
1995). Recently, however, some elegant studies of Dietschy
and coworkers have suggested that NPC1-deficient brains
undergo a progressive loss of myelin which contains a high
content of cholesterol (Xie et al. 2000; German et al. 2002).
Xie et al. also found that the cholesterol content of brains of
one-day-old npc–/– mice was higher than of npc+/+ mice (Xie
et al. 2000). Since myelin cholesterol comprises only a
negligible percentage of total brain cholesterol in neonatal
mice the authors concluded that cholesterol accumulates in
NPC1-deficient neurons and/or glial cells of the central
nervous system, as in other cell types (Xie et al. 2000).
Consequently, only in older npc–/– mice, in which extensive
demyelination in the brain has occurred, is there a net
decrease of brain cholesterol, compared to wild-type mice.
We show that npc–/– sympathetic neurons display a bright
punctate pattern of filipin fluorescence, similar to that in
NPC1-deficient fibroblasts and CHO cells (Sokol et al. 1988;
Dahl et al. 1992; Coxey et al. 1993). Filipin-positive vesi-
cles also accumulated when wild-type neurons were incuba-
ted with LDLs in the presence of progesterone or U18666A,
as has been reported for fibroblasts (Butler et al. 1992;
Kobayashi et al. 1999). In contrast, direct measurement of
cholesterol mass revealed that the cholesterol content of
npc–/– and npc+/+ neurons was the same. This apparent
discrepancy was resolved when we measured the mass of
cholesterol in cell bodies/proximal axons and distal axons
separately. We found that cholesterol is redistributed in npc–/–
neurons so that the amount of cholesterol in cell bodies of
npc–/– neurons is greater, whereas the amount in distal axons
is less, than in npc+/+ neurons. These observations are
consistent with those of Henderson et al. (Henderson et al.
2000) who found no difference between the cellular choles-
terol content of npc–/– and npc+/+ embryonic striatal neurons.
However, their study did not distinguish between cell bodies
and axons and did not report whether or not filipin-positive
vesicles accumulated in npc–/– striatal neurons.
Several studies have suggested that the cholesterol content
of the plasma membrane of npc–/– and npc+/+ fibroblasts is
the same (Lange et al. 2000; Garver et al. 2002; Lange et al.
2002) whereas other evidence has indicated that the plasma
membrane of NPC1-deficient fibroblasts (Koike et al. 1998;
Sokol et al. 1988) and CHO cells (Dahl et al. 1992) is
cholesterol-poor relative to that of wild-type cells. Our
observations are consistent with the idea that NPC1-defici-
ency decreases the content of cholesterol in plasma mem-
branes of axons since the amount of plasma membrane
relative to internal membranes is higher in axons than in
cell bodies due to the elongated shape of the neuron.
The observed alteration in the partitioning of cholesterol
between cell bodies/proximal axons and distal axons of

1162 B. Karten et al.
NPC1-deficient neurons leads us to speculate that the
anterograde movement of cholesterol from cell bodies to
distal axons is impaired in NPC1-deficient neurons.
Origin of cholesterol accumulating in npc–/– cell bodies
In npc–/– fibroblasts, cholesterol-laden vesicles are more
evident when the cells are incubated with LDLs (Sokol et al.
1988). Prolonged incubation of these cells in medium
containing delipidated serum leads to a reduction in the size
and number of these vesicles suggesting that LDLs are the
primary source of the accumulating cholesterol. In contrast,
we found that when sympathetic neurons were cultured for

2 weeks in lipoprotein-free medium, the intense punctate
pattern of filipin fluorescence remained. Even 1-day-old
npc–/– sympathetic neurons that had not been exposed to any
exogenous cholesterol during culture were filled with
cholesterol-laden vesicles, implying that these vesicles had
accumulated during gestation. Thus, our data indicate that the
cholesterol accumulating in cell bodies of npc–/– sympathetic
neurons is primarily derived from endogenous synthesis
rather than from lipoproteins. This conclusion is consistent
with the idea that neurons rely heavily on cholesterol
synthesized endogenously. Our laboratory has previously
shown that in sympathetic neurons cholesterol is synthesized
in cell bodies/proximal axons, but not in distal axons (Vance
et al. 1994; Posse De Chaves et al. 2000). An implication of
these results is that the transport of endogenously synthesized
cholesterol into distal axons of NPC1-deficient neurons
might be defective. The use of compartmented cultures of
neurons will permit us to test this hypothesis. As npc–/–
neurons survive and grow normally, at least under the culture
conditions we used, it is unlikely that NPC1 deficiency
results in a generalized defect in vesicle trafficking. Rather,
we suggest that the anterograde transport of the vehicles that
carry cholesterol is specifically impaired.
Acknowledgements
This work was supported by grants from the Canadian Institutes for
Health Research and the Ara Parseghian Medical Research
Foundation. We thank Susanne Lingrell and Russ Watts for their
excellent technical assistance and Dr G. Francis (University of
Alberta) for providing human lipoproteins.
References
Butler J. D., Blanchette-Mackie E. J., Goldin E., O’Neill R. R., Carstea
G., Roff C. F., Patterson M. C., Patel S., Comly M. E., Cooney A.,
Vanier M. T., Brady R. O. and Pentchev P. G. (1992) Progesterone
blocks cholesterol translocation from lysosomes. J. Biol. Chem.
267, 23797–23805.
Cadigan K. M., Spillane D. M. and Chang T.-Y. (1990) Isolation and
characterization of chinese hamster ovary cell mutants defective in
intracellular low density lipoprotein-cholesterol trafficking. J. Cell
Biol. 110, 295–308.
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1154–1163
14714159, 2002, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2002.01220.x by University Of British Columbia, Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Campenot R. B. (1979) Independent control of the local environment of
somas and neurites. Methods Enzymol. 28, 302–307.
Campenot R. B. (1992) Compartmented culture analysis of nerve growth,
in Cell–Cell Interactions: a Practical Approach (Stevenson R. B.,
Gallin W. J. and Paul D. L., eds), pp. 275–298. IRL Press, Oxford.
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., Nagle J., Polymeropoulos M. H., Sturley S. L., Ioannou
Y. A., Higgins M. E., Comly M., Cooney A., Brown A., Kaneski
C. R., Blanchette-Mackie E. J., Dwyer N. K., Neufeld E. B., Chang
T.-Y., Liscum L., Strauss J. F., Ohno K., Zeigler M., Carmi R.,
Sokol J., Markie D., O’Neill R. R., van Diggelen O. P., Elleder M.,
Patterson M. C., Brady R. O., Vanier M. T., Pentchev P. G. and
Tagle D. A. (1997) Niemann–Pick C1 disease gene: homology to
mediators of cholesterol homeostasis. Science 277, 228–231.
Cham B. E. and Knowles B. R. (1976) A solvent system for delipidation
of plasma or serum without protein precipitation. J. Lipid Res. 17,
176–181.
Chen J. W., Murphy T. L., Willingham M. C., Pastan I. and August J. T.
(1985) Identification of two lysosomal membrane glycoproteins.
J. Cell Biol. 101, 85–95.
Coxey R. A., Pentchev P. G., Campbell G. and Blanchette-Mackie E. J.
(1993) Differential accumulation of cholesterol in Golgi compart-
ments of normal and Niemann–Pick type C fibroblasts incubated
with LDL: a cytochemical freeze-fracture study. J. Lipid Res. 34,
1165–1176.
Cruz J. C. and Chang T.-Y. (2000) Fate of endogenously synthesized
cholesterol in Niemann–Pick type C1 cells. J. Biol. Chem. 275,
41309–41316.
Dahl N. K., Reed K. L., Daunais M. A., Faust J. R. and Liscum L. (1992)
Isolation and characterization of Chinese hamster ovary cells
defective in the intracellular metabolism of low density lipoprotein-
derived cholesterol. J. Biol. Chem. 267, 4889–4896.
Danik M., Champagne D., Petit-Turcotte C., Beffert U. and Poirier J.
(1999) Brain lipoprotein metabolism and its relation to neuro-
degenerative disease. Crit. Rev. Neurobiol. 13, 357–407.
Elleder M., Jirasek A., Smid F., Ledvinova J. and Besley G. T. N. (1985)
Niemann–Pick disease type C. Study on the nature of the cerebral
storage process. Acta Neuropathol. 66, 325–336.
Garver W. S., Krishnan K., Gallagos J. R., Michikawa M., Francis G. A.
and Heidenreich R. A. (2002) Niemann–Pick C1 protein regulates
cholesterol transport to the trans-Golgi network and plasma
membrane caveolae. J. Lipid Res. 43, 579–589.
German D. C., Quintero E. M., Liang C. L., Ng B., Punia S., Xie C. and
Dietschy J. M. (2001) Selective neurodegeneration, without neu-
rofibrillary tangles, in a mouse model of Niemann–Pick C disease.
J. Comp. Neurol. 433, 415–425.
German D. C., Liang C. L., Song T., Yazdani U., Xie C. and Dietschy
J. M. (2002) Neurodegeneration in the Niemann–Pick C mouse:
glial involvement. Neuroscience 109, 437–450.
Goodrum J. F. and Pentchev P. G. (1997) Cholesterol reutilization during
myelination of regenerating PNS axons is impaired in Niemann–
Pick disease type C mice. J. Neurosci. Res. 49, 389–392.
Goodrum J. F., Brown J. C., Fowler K. A. and Bouldin T. W. (2000)
Axonal regeneration, but not myelination, is partially dependent on
local cholesterol reutilization in regenerating nerve. J. Neuro-
pathol. Exp. Neurol. 59, 1002–1010.
Hawrot E. and Patterson P. H. (1979) Long-term culture of dissociated
sympathetic neurons. Methods Enzymol. 58, 574–584.
Henderson L. P., Lin L., Prasad A., Paul C. A., Chang T. Y. and Maue
R. A. (2000) Embryonic striatal neurons from Niemann–Pick type
C mice exhibit defects in cholesterol metabolism and neurotrophin
responsiveness. J. Biol. Chem. 275, 20179–20187.

Higashi Y., Murayama S., Pentchev P. G. and Suzuki K. (1993) Cere-
bellar degeneration in the Niemann–Pick type C mouse. Acta
Neuropathol. 85, 175–184.
Karten B. L. M. B., Eng H., Azumaya Y., Martin G., Lund K., Watts
R. C., Vance J. E., Vance D. E. and Campenot R. B. (2002)
Analytical approaches for investigating apoptosis and other
biochemical events in compartmented cultures of sympathetic
neurons, in Apoptosis Methods and Techniques, Vol. 37 (LeBlanc
A. C., ed.), pp. 163–175. Humana Press, Totowa, NJ.
Kobayashi T., Beuchat M. H., Lindsay M., Frias S., Palmiter R. D.,
Sakuraba H., Parton R. G. and Gruenberg J. (1999) Late endo-
somal membranes rich in lysobisphosphatidic acid regulate cho-
lesterol transport. Nat. Cell Biol. 1, 113–118.
Koike T., Ishida G., Taniguchi M., Higaki K., Ayaki Y., Saito M.,
Sakakihara Y., Iwamori M. and Ohno K. (1998) Decreased mem-
brane fluidity and unsaturated fatty acids in Niemann–Pick disease
type C fibroblasts. Biochim. Biophys. Acta 1406, 327–335.
Lange Y. Ye J., Rigney M. and Steck T. (2000) Cholesterol movement in
Niemann–Pick type C cells and in cells treated with amphiphiles.
J. Biol. Chem. 275, 17468–17475.
Lange Y. Ye J., Rigney M. and Steck T. L. (2002) Dynamics of lyso-
somal cholesterol in Niemann–Pick type C and normal human
fibroblasts. J. Lipid Res. 43, 198–204.
Lange Y., Ye J. and Steck T. L. (1998) Circulation of cholesterol between
lysosomes and the plasma membrane. J. Biol. Chem. 273, 18915–
18922.
Liscum L. and Faust J. R. (1987) Low density lipoprotein (LDL)-
mediated suppression of cholesterol synthesis and LDL uptake is
defective in Niemann–Pick type C fibroblasts. J. Biol. Chem. 262,
17002–17008.
Liscum L. and Faust J. R. (1989) The intracellular transport of low
density lipoprotein-derived cholesterol is inhibited in chinese
hamster ovary cells cultured with 3-b-(2-(diethylamino)eth-
oxy)androst-5-en-17-one. J. Biol. Chem. 264, 11796–11806.
Liscum L., Ruggiero R. M. and Faust J. R. (1989) The intracellular
transport of low density lipoprotein-derived cholesterol is defective
in Niemann–Pick type C fibroblasts. J. Cell Biol. 108, 1625–1636.
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., Pentchev
P. G. and Pavan W. J. (1997) Murine model of Niemann–Pick C
disease: mutation in a cholesterol homeostasis gene. Science 277,
232–235.
MacInnis B. L. and Campenot R. B. (2002) Retrograde support of
neuronal survival without retrograde transport of nerve growth
factor. Science 295, 1536–1539.
Maziere C., Maziere J. C., Mora L., Lageron A., Polonovski C. and
Polonovski J. (1987) Alterations in cholesterol metabolism in
cultured fibroblasts from patients with Niemann–Pick disease type
C. J. Inher. Metab. Dis. 10, 339–346.
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.
Pentchev P. G., Vanier M. T., Suzuki K. and Patterson M. C. (1995)
Niemann–Pick disease type C: a cellular cholesterol lipidosis, in
The Metabolic and Molecular Basis of Inherited Disease, Vol. II.
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1154–1163
14714159, 2002, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2002.01220.x by University Of British Columbia, Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Cholesterol accumulation in npc–/– neurons 1163
(Scriver C. R., Beaudet A. L., Sly W. S. and Valle D., eds), pp.
2625–2639. McGraw-Hill, New York.
Posse De Chaves E. I., Vance D. E., Campenot R. B., Kiss R. S. and
Vance J. E. (2000) Uptake of lipoproteins for axonal growth of
sympathetic neurons. J. Biol. Chem. 275, 19883–19890.
Prasad A., Fischer W. A., Maue R. A. and Henderson L. P. (2000)
Regional and developmental expression of the NPC1 mRNA in the
mouse brain. J. Neurochem. 75, 1250–1257.
Roff C. F., Goldin E., Comly M. E., Blanchette-Mackie J., Cooney A.,
Brady R. O. and Pentchev P. G. (1992) Niemann–Pick type-C
disease: deficient intracellular transport of exogenously derived
cholesterol. Am. J. Med. Genet. 42, 593–598.
Sattler W., Mohr D. and Stocker R. (1994) Rapid isolation of lipopro-
teins and assessment of their peroxidation by high-performance
liquid chromatography postcolumn chemiluminescence. Methods
Enzymol. 233, 469–489.
Sofroniew M. V., Howe C. L. and Mobley W. C. (2001) Nerve growth
factor signaling, neuroprotection, and neural repair. Annu. Rev.
Neurosci. 24, 1217–1281.
Sokol J., Blanchette-Mackie E. J., Kruth H. S., Dwyer N. K., Amende
L. M., Butler J. D., Robinson E., Patel S., Brady R. O., Comly
M. E., Vanier M. T. and Pentchev P. G. (1988) Type C Niemann–
Pick disease. Lysosomal accumulation and defective intracellular
mobilization of low density lipoprotein cholesterol. J. Biol. Chem.
263, 3411–3417.
Taniguchi M., Shinoda Y., Ninomiya H., Vanier M. T. and Ohno K.
(2001) Sites and temporal changes of gangliosides GM1/GM2
storage in the Niemann–Pick disease type C mouse brain. Brain
Dev. 23, 414–421.
Turley S. D., Burns D. K., Rosenfeld C. R. and Dietschy J. M. (1996)
Brain does not utilize low density lipoprotein-cholesterol during
fetal and neonatal development in the sheep. J. Lipid Res. 37,
1953–1961.
Turley S. D., Burns D. K. and Dietschy J. M. (1998) Preferential util-
ization of newly synthesized cholesterol for brain growth in neo-
natal lambs. Am. J. Physiol. 274, E1099–E1105.
Vance J. E., Pan D., Campenot R. B., Bussiere M. and Vance D. E.
(1994) Evidence that the major membrane lipids, except choles-
terol, are made in axons of cultured rat sympathetic neurons.
J. Neurochem. 62, 329–337.
Vanier M. T. (1999) Lipid changes in Niemann–Pick disease type C
brain: personal experience and review of the literature. Neurochem.
Res. 24, 481–489.
Vanier M. T. and Suzuki K. (1998) Recent advances in elucidating
Niemann–Pick C disease. Brain Pathol. 8, 163–174.
Xie C., Turley S. D., Pentchev P. G. and Dietschy J. M. (1999) Cho-
lesterol balance and metabolism in mice with loss of function of
Niemann–Pick C protein. Am. J. Physiol. 276, E336–E344.
Xie C., Burns D. K., Turley S. D. and Dietschy J. M. (2000) Cholesterol
is sequestered in the brains of mice with Niemann–Pick type C
disease but turnover is increased. J. Neuropathol. Exp. Neurol. 59,
1106–1117.
Zervas M., Dobrenis K. and Walkley S. U. (2001) 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.