Cholesterol accumulates in cell bodies, but is decreased in distal axons, of Niemann–Pick C1-deficient neurons


  • Barbara Karten,

    1. Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids and Departments of
    2. Medicine,
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    • 1

      Supported by postdoctoral fellowships from the Alberta Heritage Foundation for Medical Research and the Deutsche Forschungsgemeinschaft (Forschungsstipendium KA1578/1).

  • Dennis E. Vance,

    1. Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids and Departments of
    2. Biochemistry and
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    • 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.

  • Robert B. Campenot,

    1. Cell Biology, University of Alberta, Edmonton, Alberta, Canada
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    • 2

      Medical Scientists of the Alberta Heritage Foundation for Medical Research.

  • Jean E. Vance

    1. Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids and Departments of
    2. Medicine,
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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:


Niemann–Pick type-C (NPC) disease is characterized by a progressive loss of neurons and an accumulation of unesterified cholesterol within the endocytic pathway. Unlike other tissues, however, NPC1-deficient brains do not accumulate cholesterol but whether or not NPC1-deficient neurons accumulate 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, 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.

Abbreviations used

high density lipoprotein


lysosome-associated membrane protein 1


low-density lipoprotein


nerve growth factor


Niemann–Pick type C


phosphate-buffered saline


sodium dodecyl sulfate



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, meganeurites 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 fibroblasts and mutant CHO cells (Cadigan et al. 1990; Liscum 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; 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 (Taniguchi 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 cholesterol 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


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 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 (Lexington, 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-β-(2-diethylaminoethoxy)androst-5-en-17-one] was purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Other materials for gel electrophoresis and immunoblotting were from Bio-Rad (Mississauga, ON, Canada). Monoclonal antiβ-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. Delipidated 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, originally 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 compartments (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 center compartment and 100 ng/mL NGF for the side compartments. During the first week after plating, 10 µm 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 ‘axotomy’) 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 β-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-β-tubulin antibody (dilution 1 : 1000). Immunoblots 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 consisting 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 (ECL Western 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 µL 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 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT tetrazolium salt) into an insoluble formazan product using the CellTiter 96® 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 µg/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 µCi/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, 5α-cholestane, was added. Unesterified cholesterol was converted to its trimethylsilyl ether derivative by reaction with bis(trimethylsilyl)trifluoroacetamide 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).


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).

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 immunoreactive 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).

Figure 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.

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 differences 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 genotypes we demonstrated by immunoblotting that the amount of β-tubulin, the most abundant cytoskeletal protein of axons, was the same in distal axons of npc+/+, npc+/– and npc–/– neurons (Fig. 2b).

Figure 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. ○, npc+/+; □, npc+/–; ▵, 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 electrophoresis and immunoblotting with antiβ-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.

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 compartmented 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.

Figure 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 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 cytoplasmic 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).

Figure 4.

Cholesterol-filled vesicles accumulate in cell bodies of npc–/– neurons. Sympathetic 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 = 20 µm. The experiment was performed four times with similar results.

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 colocalization 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 accumulates 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. accumulation of LDL-derived cholesterol in intracellular vesicles) in fibroblasts (Liscum and Faust 1989; Butler et al. 1992). To determine if these drugs also caused an accumulation 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).

Figure 5.

Cholesterol-filled vesicles accumulate 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 µm progesterone and 0.1 mg/mL LDLs (b), 1.5 µm U18666A and 0.1 mg/mL LDLs (c), 20 µm progesterone (d), or 1.5 µm U18666A (e). Filipin staining was examined by confocal microscopy. Scale bar = 20 µm. The experiment was performed three times with similar results.

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 (µg cholesterol/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 absence of filipin. A punctate fluorescence pattern was observed in the UV excitation range at 351 nm, similar to, but of much lower intensity (∼5%) than, the filipin staining in Fig. 4. Therefore, the autofluorescence exhibited by npc–/– neurons is only a minor contributor to the high fluorescence intensity observed after filipin staining.

Figure 6.

Cholesterol content and distribution in sympathetic neurons. Sympathetic npc+/+, npc+/–, and npc–/– neurons were maintained for 12 days in 48-well plates (a) or compartmented culture dishes (b). Lipids were extracted and the cholesterol content measured. In (a) values are given as µg cholesterol/mg protein and are means ± SE of three independent experiments with between four and six wells in each experiment. The cholesterol content of distal axons and cell bodies/proximal axons (b) is given as percentage of the total cholesterol content of cell bodies/proximal axons plus distal axons. Data are averages ± SE of five independent experiments. *p < 0.00075 for npc–/– versus npc+/+ neurons.

We next considered the possibility that the reason that the cholesterol content of npc–/– neurons as a whole was not increased (Fig. 6a) was because the accumulation of cholesterol in cell bodies/proximal axons was compensated by a reduced amount of cholesterol in distal axons. We therefore measured the cholesterol content of cell bodies/proximal 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 cholesterol 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 µg/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.

Figure 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.

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 determine whether or not the formation of cholesterol-laden vesicles 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 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.

Figure 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 and examined by confocal microscopy. (a) and (d) npc+/+, (b) and (e ) npc+/–, (c) and (f) npc–/–. Scale bar = 20 µm. The experiment was repeated with similar results.

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.


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 retrograde 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 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 vesicles also accumulated when wild-type neurons were incubated 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 cholesterol 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-deficiency decreases the content of cholesterol in plasma membranes 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 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.


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.