Trafficking of Cholesterol from Cell Bodies to Distal Axons in Niemann Pick C1-deficient Neurons*

Barbara KartenDagger §, Dennis E. VanceDagger ||**DaggerDagger, Robert B. Campenot**§§, and Jean E. VanceDagger §¶¶

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -cyclodextrin (Mbeta 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.

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-/- 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.

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 Mbeta 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 Mbeta CD/cholesterol with a Mbeta CD:cholesterol molar ratio of 10:1 and a specific radioactivity of 10 mCi/mmol Mbeta CD. Directly before use, the solution was filter-sterilized to remove microcrystals of cholesterol not complexed with Mbeta CD, resulting in a final specific radioactivity of 6.6 mCi/mmol Mbeta CD.

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 Mbeta 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/Mbeta 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.

Statistical Analyses-- The statistical significance of differences (p < 0.05) was determined using the Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of NPC1 Protein in Murine Sympathetic Neurons-- Sympathetic neurons from npc+/+, npc+/-, 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).

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-/- 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.

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.


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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.

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-/- 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 Mbeta 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 Mbeta 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 Mbeta 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.

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-/- 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.

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 (Mbeta CD). ApoE-free HDLs containing [3H]cholesteryl oleate or Mbeta 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 Mbeta 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).

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.


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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.

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-/- 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.

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-/- 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).

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).


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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.

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.

                              
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Table I
Phenotypes elicited in sympathetic neurons by progesterone U18666A, and a lack of functional NPC1


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-/- 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.

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 Mbeta 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).

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-/- 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.

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-/- 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.

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-/- 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.

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-/- 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.

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-/- neurons and in wild-type neurons exposed to U18666A, LDL cholesterol is not delivered to the site required for axonal growth.

Conclusions-- Our experiments show that LDL cholesterol is transported into axons of npc-/- 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.

Dagger Dagger 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; Mbeta CD, methyl beta -cyclodextrin; NGF, nerve growth factor; PBS, phosphate-buffered saline; U18666A, 3-beta -[2-diethylaminoethoxy]androst-5-ene-17-one.

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ABSTRACT
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RESULTS
DISCUSSION
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