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Originally published In Press as doi:10.1074/jbc.M405345200 on September 7, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48654-48662, November 19, 2004
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Secretion of Sterols and the NPC2 Protein from Primary Astrocytes*

Aino-Liisa Mutka{ddagger}, Sari Lusa{ddagger}, Matts D. Linder§, Eija Jokitalo§, Outi Kopra{ddagger}, Matti Jauhiainen{ddagger}, and Elina Ikonen§

From the §Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014 University of Helsinki and {ddagger}National Public Health Institute, Biomedicum Helsinki, Haartmaninkatu 8, 00251 Helsinki, Finland

Received for publication, May 13, 2004 , and in revised form, August 25, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1–/–) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1–/– astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The brain is the most cholesterol-rich organ in the body, containing roughly 25% of the unesterified cholesterol present in the whole individual. The input of cholesterol into the brain comes entirely, or almost entirely, from in situ synthesis because blood lipoproteins do not cross the blood-brain-barrier (1). Glial cells are thought to be responsible for a large part of this biosynthetic activity. Most of the sterol in the brain is acquired during myelination in the early stages of development and is produced by oligodendrocytes (2).

Astrocytes, the most abundant glial cells, are intimately associated with neuronal synapses and secrete cholesterol in lipoprotein particles. Astrocytes have been proposed to provide cholesterol for synapse formation (3) as well as to participate in the recycling of cholesterol after injury (4). Nascent lipoproteins isolated from neonatal mouse astrocytes are a heterogeneous population of particles in the size range of plasma high density lipoproteins and appear to be composed of two separate classes that contain either apolipoprotein E or J (5).

Niemann-Pick type C disease (NPC)1 is an inherited, fatal neurodegenerative disorder in which large amounts of cholesterol and sphingolipids accumulate intracellularly within late endocytic organelles. The disease is caused by mutations in either the NPC1 or NPC2 gene (6). The NPC1 protein is a late endosomal membrane protein harboring a sterol-sensing domain, whereas NPC2 is a secretory protein that has cholesterol binding properties and uses the mannose 6-phosphate marker for late endosomal targeting (7). The exact functions of the proteins remain unknown, but genetic evidence suggests that they function in concert to facilitate lysosomal lipid egress (8). In the NPC1–/– mouse astrocytes are considered to contribute to neurodegeneration (9, 10). Interestingly, NPC1–/– astrocytes were shown to have defects in the trafficking of endogenously synthesized cholesterol (11).

We have previously analyzed cholesterol biosynthesis and sterol efflux to extracellular acceptors in non-neuronal cells. In both fibroblastic and hepatic cells, the individual precursor sterols identified were released differentially from each other and from cholesterol (12). In the present study, we analyzed the synthesis and secretion of cholesterol and late intermediates of cholesterol biosynthesis in wild-type (WT) and NPC1-deficient murine astrocytes. We show that specific cholesterol precursors represent major sterol species both within astrocytes and in astrocyte-derived sterol-carrying particles. Moreover, we provide evidence suggesting that the majority of cholesterol is not secreted together with NPC2 and that the release of neither sterols nor NPC2 depends on NPC1 function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Diethyl ether and petroleum ether were from J. T. Baker Inc.; all other HPLC-grade solvents, lovastatin, and the silica gel 60 TLC plates were from Merck. [4-14C]Cholesterol (specific activity 58 mCi/mmol), [1{alpha},2{alpha}(n)-3H]cholesterol (specific activity 41.0 Ci/mmol), [3H]acetic acid (specific activity 5.10–9.00 Ci/mmol), Hybond-C membrane, and the ECL reagents were purchased from Amersham Biosciences. The polyclonal anti-glial fibrillary acidic protein antibody was from Dako, and anti-lysobisphosphatidic acid antibody (13) was a gift from Jean Gruenberg (University of Geneva). The polyclonal anti-NPC2 antibody has been described (14). Endoglycosidase H and peptide N-glycosidase F were from New England Biolabs. Poly-D-lysine, Dulbecco's modified Eagle's medium, filipin, mevalonic acid lactone (mevalonate), and protease inhibitors were from Sigma. The G-5 supplement was from Invitrogen, and the characterized fetal bovine serum was from HyClone.

Animals—Balb/c control mice were obtained from Harlan (Horst, The Netherlands). Balb/c mice heterozygous for the NPC1 mutation were provided by the Pentchev laboratory at the National Institutes of Health (15). The mice were allowed food and water ad libitum and kept at a 12-h light-dark cycle, 22 °C temperature, and 50% relative air humidity. Mice heterozygous for the NPC1 mutation were used to generate the NPC1–/– embryos. The genotypes of individual embryos were determined from genomic DNA obtained from tissue samples using the polymerase chain reaction and oligonucleotides as described (15).

Cell Culture—Primary astrocyte cultures from E16.5 embryos were prepared and cultured essentially as described (16). Briefly, astrocytes prepared from the striata of the embryos were cultured in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.5 mM L-glutamine, 25 mM Hepes, pH 7.4, 1x G-5 supplement, and 1% characterized fetal bovine serum on poly-D-lysine (50 µg/ml)-coated culture dishes (Nunc). The cells were cultured at 37 °C under 5% CO2 and 95% air for 7–12 days during which they were passaged 1–3 times before the experiments. At least 85% of the cells were astrocytes as judged by the percentage of anti-glial fibrillary acidic protein-positive cells. Primary fibroblasts (F92–99) and their culture conditions have been described (17).

Radiolabeling of Cells—The cells were seeded on 55-mm poly-D-lysine-coated culture dishes or 75-cm2 vessels in 1% characterized fetal bovine serum medium. The following day cells were washed a minimum of 6 times with PBS, and the growth medium was replaced with serum-free culture medium containing [14C]cholesterol (30–100 nCi/ml, 3 ml/dish). After 24–48 h of culture, the cells were washed with PBS, labeled with [3H]acetate (250–500 µCi/ml, 1.5 ml/dish) for 60 min at 37 °C, and chased at 37 °C for 2 or 18 h as indicated in serum-free culture medium in the presence of 10 µM lovastatin and 25 mM mevalonate. This labeling scheme is referred to as A. For equilibrium labeling, the cells were incubated with 1–2 µCi/ml [3H]cholesterol in culture medium for 2 days. The cells were then washed 6 times with PBS and seeded on 75-cm2 culture vessels in medium containing 1% (v/v) lipoprotein-deficient serum prepared as described (18) and 1–2 µCi/ml [3H]cholesterol. After 2 days of culture, the cells were washed several times with PBS and chased overnight in fresh, non-labeled serum-free culture medium, 7 ml/vessel. This labeling scheme is referred to as B. Identical treatment without the radiolabel is referred to as C.

Analysis of Sterol Biosynthesis, Efflux, and Quantity—The medium and cells labeled as in A were collected, and aliquots of both were analyzed by liquid scintillation counting to determine the [14C]cholesterol activity. Lipids were extracted, separated by TLC, and analyzed by Ag+-HPLC and liquid scintillation counting as described (19). For quantitation of unlabeled sterols, the Ag+-HPLC eluent was passed through a PerkinElmer Life Sciences Series 200 UV-visible detector set at 210 nm. The instrument response was measured by comparing the peak areas of individual pure sterols (Steraloids).

Flotation of Membranes in Sucrose Gradient—Flotation of cellular membranes in sucrose gradients was performed essentially as described (20). Briefly, two confluent 75-cm2 flasks of astrocytes cultured as in C were washed twice with PBS and scraped in 250 mM sucrose, 140 mM KCl, 10 mM Hepes, pH 7.4, and broken by repeated passages through a 23-gauge needle. After removal of nuclei and intact cells, sucrose was added to a final concentration of 2 M (total volume of 2 ml). This was overlaid with 1.6 ml of 1.7 M of sucrose and 0.4 ml of 0.8 M sucrose in 140 mM KCl, 10 mM Hepes, pH 7.4. The gradients were centrifuged at 37,000 x g in a SW60Ti rotor at 10 °C for 21 h. Fractions of 0.8 ml were collected from the top, and lipids were extracted from each fraction and analyzed by TLC and Ag+-HPLC.

Size-exclusion Chromatography—Media from cells labeled as in A, B, or C and/or infected with recombinant Semliki Forest viruses as indicated were collected and centrifuged at +4 °C (2500 rpm, 10 min) to remove cellular debris. The media were then fractionated either after concentration by Centricon-10 filter units (Millipore) or unconcentrated using a Superose 6HR size-exclusion chromatography column (10 x 30, Amersham Biosciences). Medium (1 ml) was applied onto the column equilibrated with PBS. The flow rate was 0.5 ml/min, and 0.5-ml fractions were collected. The eluted fractions were analyzed for radioactivity by liquid scintillation counting. Where indicated, fractions were subjected to further analysis by TLC, HPLC, electron microscopy, or Western blotting as detailed elsewhere.

Recombinant Semliki Forest Virus Expression—The NPC2 cDNA (14) cloned into the HindIII-EcoRV sites of pCR3.1 was excised as a HindIII-XbaI fragment, blunted with T4 DNA polymerase, and subcloned into the SmaI site of pSFV1. pSFV-LacZ, pSFV-NPC2, and pSFV-helper 1 were linearized, and runoff transcription was performed according to Liljeström and Garoff (21). The NPC2 transcription mix was cotransfected with the helper transcription mix into baby hamster kidney cells using electroporation as described (22). The culture supernatant was collected after 24 h of incubation. Titration of the viral stocks on baby hamster kidney cells was performed as described (22). Recombinant SFV was added on near-confluent dishes of astrocytes, and viral entry was allowed for 60 min at 37 °C, after which the incubation was continued in serum-free astrocyte medium at 37 °C for 6 or 14 h (latter for size-exclusion chromatography).

Endoglycosidase Digestion and Western Blotting—For endoglycosidase digestion, cells were lysed in 1% Nonidet-P40 in PBS supplemented with protease inhibitors (chymostatin, leupeptin, antipain, and pepstatin at 25 µg/ml each). Equal amounts (20 µg) of protein were incubated with endoglycosidase H or peptide N-glycosidase F according to manufacturer's instructions. For immunoblotting medium after size-exclusion chromatography, pooled fractions were dialyzed against water overnight using Spectra/Por dialysis membrane molecular weight cut-off 3500 (Spectrum Laboratories), lyophilized, and resuspended in Laemmli sample buffer. For analyzing NPC2 secretion, cells were changed to fresh serum-free medium (1 ml/dish), and cells and media were harvested after 6 h. Aliquots of media (0.69–1 ml/dish, amount adjusted for total protein, 113–163 µg/dish) were concentrated using Centricon YM-10 filter units (Millipore) and separated together with cell lysates (10 µg protein) by 15% SDS-PAGE. Proteins were transferred to Hybond-C Extra membrane, blocked with 3% defatted bovine serum albumin in Tris-buffered saline, 0.1% Tween 20, and immunoblotted using anti-NPC2 antibodies (1:1000), horseradish peroxidaseconjugated secondary antibodies, and enhanced chemiluminescence detection. The specificity of the ~22 kDa band for NPC2 was confirmed by analyzing NPC2–/– mouse tissue in which no immunoreactivity was found (data not shown). Densitometric scanning of the intensity of the NPC2 bands was performed with Advanced Image Data Analyzer (AIDA software).

Immunofluorescence and Negative Staining Electron Microscopy— Filipin and antibody staining for immunofluorescence microscopy was performed as described in Hölttä-Vuori et al. (23) under "Immunocyto-chemistry." The coverslips were viewed with a Leica TCS SP confocal microscope or an Olympus IX70 inverted microscope equipped with a Polychrome IV monochromator (TILL Photonics) with appropriate filters. For electron microscopy, medium from cells treated as in B was either stained as such or concentrated 14-fold with a Centricon-10 filter unit. The concentrated medium was subjected to size-exclusion chromatography, and material from pooled fractions 16–18 was stained. Fresh culture medium, similarly concentrated, was used as a control. Samples were adsorbed onto carbon-coated Pioloform grids and negatively stained with 2% potassium phosphotungstate, pH 7.2, before air drying. Grids were viewed using FEI Tecnai 12 electron microscope (Philips Electron Optics) at 80 kV. Spherical profiles were not found in fresh culture medium, and few profiles were observed in non-fractionated medium. For estimation of the particle diameter, pictures taken at a magnification of 49000 were scanned, and the diameter was measured with the use of the Image-Pro Plus software package, version 4.5.1.29 [EC] . Measurements were done on 268 particles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sterol Biosynthesis and Major Sterol Species in Astrocytes— Astrocytes in vivo are thought not to encounter serum lipoproteins, and their presence changes sterol balance in the cells. Both cholesterol biosynthesis and esterification are modulated by lipoproteins (24, 25). Because fully serum-free cultures did not efficiently survive and expand in vitro, the cells were cultured in the presence of 1% serum. Under these conditions, cell growth was slower than in the presence of 10–20% serum, but the cells divided and after 7–10 days displayed strong anti-glial fibrillary acidic protein (anti-GFAP) immunoreactivity (Fig. 1).



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  		FIG. 1.
Cholesterol deposits are found in NPC1–/– astrocytes. The cells were stained with filipin to visualize cellular free cholesterol and with the antibody against glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. The vast majority of cells are astrocytes. a and b, WT cells. c, in NPC1–/– cells the pathognomonic cholesterol deposits colocalize with the late endosomal marker anti-lysobisphosphatidic acid (LBPA). Scale bar, 10 µm.

 
The cells were labeled with [14C]cholesterol for 2 days and then pulse-labeled for 60 min with [3H]acetate followed by chasing for 2 or 18 h in the presence of lovastatin and mevalonate. [14C]Cholesterol labeling traces pre-existing cellular cholesterol pools, whereas newly synthesized sterols incorporate [3H]acetate. Lipids were extracted and separated by TLC followed by Ag+-HPLC to resolve individual sterol species. At 2 h of chase, desmosterol was the major biosynthetic [3H]sterol identified and was considerably more abundant than newly synthesized [3H]cholesterol. Cholesterol, zymosterol, and lathosterol represented minor radiolabeled sterol species (Fig. 2, a and c). After overnight chasing, the fraction of [3H]cholesterol increased, but unexpectedly, there was still a considerable fraction of [3H]desmosterol present (Fig. 2, b and c). These data suggest that the conversion of the immediate precursor of cholesterol to the end product was inefficient. We, therefore, analyzed the sterol composition of unlabeled astrocytes. The HPLC analysis revealed that desmosterol was roughly as abundant as cholesterol (cholesterol:desmosterol ratio = 1.00:1.18), whereas lathosterol and zymosterol represented minor sterol species in the cells (Fig. 2d).



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  		FIG. 2.
Wild-type astrocytes synthesize cholesterol and partially retain sterol precursors. Cells were labeled with [14C]cholesterol for 48 h, pulsed with [3H]acetate for 60 min, and chased in serum-free medium. Lipids were extracted and analyzed by TLC and Ag+-HPLC. Representative HPLC profiles froma2(a) and 18 h (b) chase are shown. At 2 h, most of the [3H]activity is not converted to cholesterol. [3H]Desmosterol remains a major sterol after 18 h. Rates of synthesis from [3H]acetate for each sterol were corrected for protein amounts (c). Values represent average ± S.E. (n = 3–4) from a representative experiment. Unlabeled astrocytes cultured in a similar manner were collected, and lipids were extracted and analyzed by TLC and Ag+-HPLC using UV detection (d). chol, cholesterol; desmo, desmosterol; latho, lathosterol; zymo, zymosterol; mAU, milliabsorbance units.

 
To rule out that the sterol composition was affected by the in vitro culture conditions, we analyzed the sterol profile in freshly prepared striata immediately after the dissociation of the cells. Also in this preparation, desmosterol was of similar abundance as cholesterol (data not shown). To study whether desmosterol is a constituent of astrocyte membranes, a membrane fraction was isolated by flotation in a sucrose gradient, and the ratio or cholesterol and desmosterol was compared. In the membranes with equilibrium density between 0.8 and 1.7 M sucrose, the cholesterol:desmosterol ratio was similar to that in the total cell extract. The result indicates that desmosterol is a major astrocyte membrane sterol, being roughly as abundant as cholesterol.

Secretion of Sterols from Astrocytes—The amount of total [3H]-radiolabeled sterols in cells decreased upon chasing (Fig. 2c), suggesting that at least some of the sterols were secreted. We, therefore, analyzed the appearance of [3H]sterols in the culture medium at the same time points as in Fig. 2c, i.e. at 2 and 18 h of chase. Increasing amounts of all the biosynthetic sterols detected were recovered from the culture medium with increasing chase time (Fig. 3a). The efflux of lathosterol was most efficient, with more than 50% of the radiolabeled pool recovered from the medium after overnight chasing (Fig. 3b). The high efflux percentage is partially due to the low [3H]lathosterol levels in cells (see Fig. 2c). The efflux of [3H]desmosterol paralleled most closely with that of [3H]cholesterol, in line with previous results (12).



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  		FIG. 3.
Wild-type astrocytes secrete cholesterol and its precursors in the absence of extracellular acceptors. Cells were labeled with [14C]cholesterol for 48 h, pulsed with [3H]acetate for 60 min, and chased in serum-free medium. Lipids were extracted and analyzed by TLC and Ag+-HPLC. Amount of radiolabeled sterols in the medium increased as a function of time (a). chol, cholesterol; desmo, desmosterol; latho, lathosterol; zymo, zymosterol. Different sterols were secreted with differential efficiency, the efflux of lathosterol being the most efficient (b). Values represent average ± S.E. (n = 3–4) from a representative experiment. Similarly labeled medium (chased for 18 h) was analyzed by size exclusion chromatography (c), fractions were pooled (16–20, 21–25, etc.), and lipids were extracted and analyzed by TLC and Ag+-HPLC. Nearly all biosynthetic sterols and [14C]cholesterol was found in fractions 16–20 (d).

 
To assess the size of the particles which the radiolabeled sterols associated with in the medium, the medium was analyzed by gel filtration on a Superose 6HR column. The radiolabeling protocol was the same as in Fig. 2, and the medium was collected after overnight chasing. The majority of the pre-labeled [14C]cholesterol was recovered in fractions 16–20 (Fig. 3c), and the bulk of the [3H]acetate-derived radioactivity appeared in fractions corresponding to or after the smallest standard (1350 Da). To resolve the [3H]acetate-derived sterols, pools of 5 fractions (16–20, 21–25, etc) were extracted, and lipids were analyzed by HPLC. We found that practically all of the [3H]acetate-derived sterols effluxed from cells were recovered from fractions 16–20 together with [14C]cholesterol (Fig. 3d).

The cholesterol-containing fractions were then pooled, concentrated by using a Centricon-10 filter, and analyzed by negative staining electron microscopy. This revealed heterogeneous spherical particles (Fig. 4a) ranging in diameter from <15 nm to >30 nm, with an average diameter of 20 nm. The size distribution of the particles is given in Fig. 4a. HPLC analysis of the unlabeled sterol species in the culture medium showed that lathosterol, cholesterol, and desmosterol were the predominant sterols (Fig. 4b). The efficient efflux of lathosterol agrees with the result obtained by analyzing radiolabeled sterols. Together, the data suggest that sterol precursors lathosterol and desmosterol are major sterols in addition to cholesterol in the particles secreted by astrocytes.



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  		FIG. 4.
Astrocytes secrete spherical particles. Medium was collected from WT astrocytes and concentrated by size-exclusion chromatography, and the peak fractions containing cholesterol were subjected to negative staining electron microscopy. Spherical, heterogeneous particles ranging from <15 to >30 nm in diameter were identified. The size distribution of the particles is shown. Scale bar, 100 nm (a). The medium was also subjected to lipid extraction and analysis by TLC, Ag+-HPLC, and UV detection. Lathosterol (latho), cholesterol (chol), and desmosterol (desmo) were the dominant sterol species (b).

 
Sterol Synthesis and Secretion in NPC1–/– Astrocytes— Next, sterol synthesis and secretion in NPC1-deficient astrocytes was analyzed. These cells displayed prominent cholesterol accumulation in late endocytic organelles as visualized by the colocalization of bright, filipin-positive deposits with anti-LPBA immunoreactivity (Fig. 1c). Cells derived from wild-type, NPC1 heterozygous, and NPC1 null mice were pre-labeled with [14C]cholesterol for 2 days and pulse-labeled for 60 min with [3H]acetate followed by chasing for 18 h. We found that sterol biosynthesis was slightly but not significantly enhanced in NPC1 heterozygous and null astrocytes as compared with WT and that the sterol profile was similar between the three genotypes (Fig. 5a). The lack of increased cholesterol synthesis in NPC1-deficient astrocytes agrees with data obtained from NPC1-deficient sympathetic neurons (26). Radiolabeled sterols were then analyzed from the chase medium, and the fractions of individual sterols in cells and medium were compared. The percentage of sterols secreted to the medium was not significantly different between the three genotypes (Fig. 5b).



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  		FIG. 5.
Secretion of de novo sterols is not dependent on NPC1 genotype. Cells were labeled with [14C]cholesterol for 48 h, pulsed with [3H]acetate for 60 min, and chased in serum-free medium for 18 h. Cells and medium were collected, and lipids were extracted and analyzed by TLC and Ag+-HPLC. In NPC1–/– and NPC1+/– astrocytes, sterol synthesis was closely similar to that in WT cells (a). chol, cholesterol; desmo, desmosterol; latho, lathosterol; zymo, zymosterol. Secretion of sterols was not significantly different between the genotypes (b). Values represent the average ± S.E. (n = 3) from a representative experiment. Unlabeled NPC1–/– cells and medium treated as in C (see "Experimental Procedures") were collected, and lipids were extracted and analyzed by TLC, Ag+-HPLC, and UV detection. Cholesterol and desmosterol were found to be the major sterols in the cells, with small amounts of lathosterol and zymosterol (c). In the medium, cholesterol and desmosterol were the dominant sterols (d). mAU, milliabsorbance units.

 
To study the total sterols in NPC1–/– astrocytes, cells and medium from unlabeled cells were collected, and lipids were separated by TLC and Ag+-HPLC. The sterol profile in both the cells and medium was very similar to that observed for WT samples. Namely, cholesterol and desmosterol were the predominant sterol species in the cells (Fig. 5c; compare with Fig. 2d) and lathosterol, cholesterol and desmosterol in the medium (Fig. 5d; compare with Fig. 4b). The result confirms the efficient efflux of lathosterol seen with radiolabeled samples and shows that the total sterol profile in NPC1–/– cells is complex but not significantly different from WT.

Secretion of Endogenous and Overexpressed NPC2 from Astrocytes—Lack of NPC2 function results in an NPC disease phenotype that in man is indistinguishable from lack of NPC1 function (6). Furthermore, the NPC2 hypomorphic mouse closely resembles the NPC1 null mouse both with respect to disease progression and with biochemical and cellular neuropathological criteria (8). NPC2 was originally identified as the major secretory protein of the epididymis (27). It is known to bind cholesterol and can be secreted from cells as cholesterol bound (28, 29). It was, therefore, of interest to analyze the secretion of NPC2 from normal and NPC1–/– astrocytes.

Human NPC2 cDNA was expressed in astrocytes using the Semliki Forest virus expression system (21). The endogenous murine NPC2 protein was visualized as a single band of ~22 kDa by Western blotting (Fig. 6a). After removal of N-glycans the polypeptide chain migrated at ~16 kDa. Most or all of the sugars were of high mannose type as indicated by the similar mobility of the protein after endoglycosidase H and F treatments (Fig. 6a). Human NPC2 migrated as a doublet slightly below 25 kDa as shown previously (30) and contained both endoglycosidase H-sensitive and -resistant glycans (Fig. 6a). The slower mobility of human NPC2 was utilized to distinguish between the endogenous and overexpressed NPC2 in astrocytes (Fig. 6a).



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  		FIG. 6.
NPC2 is glycosylated and secreted from astrocytes. Lysates from normal human fibroblasts, non-infected astrocytes, and astrocytes infected with a recombinant SFV encoding human NPC2 were incubated with either endoglycosidase H (EndoH) or peptide N-glycosidase F (PNGaseF), and analyzed by Western blotting using anti-NPC2 antibodies (a). Wild-type and NPC1–/– astrocytes and medium were collected from non-infected and NPC2/SFV-infected cells. Aliquots of cell lysates and concentrated media were analyzed by Western blotting using anti-NPC2 antibodies (b). Both endogenous and recombinant NPC2 were secreted into the medium in both NPC1–/– and NPC1+/+ astrocytes. Quantitation of endogenous NPC2 band intensities (c), demonstrating moderately increased levels of NPC2 in NPC1–/– as compared with NPC1+/+ (p = 0.02 between NPC1+/+ and NPC1–/– cells; p = 0.09 between media; n = 4, Student's t test). The level of NPC2 in NPC1+/+ samples was set as 1.0.

 
The secretion of endogenous and overexpressed NPC2 was studied by immunoblotting proteins from cells and concentrated medium without virus infection or after 6 h of infection with recombinant viruses. The endogenous levels of NPC2 were higher in NPC1–/– astrocytes as compared with WT (Fig. 6, b and c). The endogenous protein was secreted to the medium both from WT and NPC1–/– astrocytes, with the higher levels paralleled by efficient secretion from NPC1–/– cells (Fig. 6, b and c). The overexpressed NPC2 was also readily secreted from both cell types (Fig. 6b). When the relative amounts of the protein in the cells and medium were compared, the overexpressed NPC2 appeared to be secreted from the cells more effectively than the endogenous protein (Fig. 6b). This may be due at least partially to the saturation of the mannose 6-phosphate receptor targeting system upon overexpression.

To determine if NPC2 was co-secreted with the cholesterol containing particles, astrocytes were equilibrium-labeled with [3H]cholesterol, infected with recombinant SFVs, and incubated overnight in the absence of radiolabel, and the medium was collected. The medium was fractionated as above, and the fractions were used for measuring [3H]cholesterol radioactivity and for trichloroacetic acid precipitation and immunoblotting with anti-NPC2 antibodies. The vast majority of cholesterol was again recovered from fractions 16–20 (Fig. 7a). Instead, endogenous NPC2 was precipitated from pooled fractions 36–40 corresponding to the size of monomeric NPC2, as visualized from cells infected with control virus (LacZ/SFV, Fig. 7b). When the medium from cells infected with NPC2/SFV was analyzed, we found that the amount of immunoreactive NPC2 in the medium was significantly increased as expected, and the protein was recovered from the same fractions (36–40) (Fig. 7b). The amount of [3H]cholesterol radiolabel and its elution profile from the Superose column were not significantly different from those in LacZ/SFV infected samples (Fig. 7a).



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  		FIG. 7.
NPC2 is not secreted together with the major cholesterol pool. Wild type astrocytes were labeled as in B (see "Experimental Procedures") and infected with LacZ/SFV or NPC2/SFV. Medium was analyzed by size-exclusion chromatography, and fractions were pooled (15–20, 21–25, etc.), dialyzed, and then analyzed by Western blotting using anti-NPC2 antibodies. The overexpression of NPC2 caused no significant change in the elution pattern of [3H]cholesterol or an increase in its efflux (a). NPC2 was found to migrate in fractions between the 44- and 17-kDa standards (b).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study the secretion of biosynthetic sterols and the NPC2 protein from WT and NPC1-deficient embryonic astrocytes was investigated. Endo-lysosomal cholesterol deposition is an early phenomenon in NPC1-deficient neuronal cells and can be observed by filipin staining in sympathetic neurons prepared from 1-day-old mice (26, 31). This was shown not to reflect an increase in cellular cholesterol mass but, rather, a redistribution of cholesterol from distal axons to the cell bodies and was accompanied by impaired trafficking of cholesterol from the soma to distal axons (31). We found prominent endolysosomal cholesterol accumulation in astrocytes prepared from E16.5 NPC1–/– embryos (filipin-positive deposits in the cell soma were evident also in hippocampal neurons prepared from the same embryos2). Thus, subcellular cholesterol mistrafficking was already evident in NPC1-deficient neuronal and glial cells isolated from mouse embryos. It remains possible, however, that the sterol deposition was associated with the in vitro culture conditions. In brain sections, astrocyte activation and cholesterol accumulation were first observed at postnatal day 9 (32).

Astrocyte cholesterol secretion is considered to be important in the maintenance and development of neuronal functions (33). In agreement with earlier studies on astrocyte cholesterol secretion (5), we found the sterols to be released in heterogeneously sized particles with spherical appearance by electron microscopy. The slightly different size range of the particles may be due to the different culture conditions or to the different region and age of the brain tissue used as a starting material.

Recently, NPC1–/– astrocytes were shown to have impaired efflux of cholesterol to methyl-{beta}-cyclodextrin (11). We found no impairment in the secretion of biosynthetic sterols in NPC1–/– astrocytes. However, when cholesterol efflux from NPC1–/– astrocytes to a cyclodextrin acceptor was analyzed as in Reid et al. (11) a significant inhibition compared with WT cells was observed.2 Thus, cholesterol efflux from the plasma membrane to cyclodextrin was impaired, although sterol secretion was not. Interestingly, Suresh et al. (34) found that in neonatal NPC1–/– astrocytes apolipoprotein E secretion was not impaired, whereas apolipoprotein D expression was up-regulated, and secretion was reduced. The normal secretion of sterols in NPC1–/– astrocytes observed in the present study suggests that the malfunctioning of the NPC1–/– neuronal cells may not be due to the inability of astrocytes to provide sterol to sustain neuronal functions, at least not at this stage of development. Neuronal functions are, however, already disturbed as shown by the impaired neurotrophin responsiveness in E16 striatal NPC1–/– neurons (35).

The intracellular trafficking itineraries and secretion of NPC2 have not been analyzed in detail either in WT or in NPC1-deficient cells. The present results show for the first time that WT and NPC1–/– astrocytes are capable of secreting the NPC2 protein and that NPC2 is secreted separately from the majority of astrocyte-derived sterols. More detailed analyses are needed to distinguish whether NPC1 deficiency leads to subtle alterations in the routing of NPC2 in astrocytes. Considering that NPC2 binds cholesterol, the secretion of overexpressed NPC2 might modulate cholesterol secretion. Recombinant SFV-driven overexpression increased the amount of immunoreactive NPC2 in the medium at least 10-fold (see Fig. 7). However, a corresponding increase in the [3H]cholesterol radioactivity in the relevant fractions was not observed. The amount of NPC2 protein may have been too small despite the overexpression to introduce a [3H]cholesterol signal to exceed the background radioactivity as the stoichiometry of NPC2 and sterol is apparently 1:1 (36). Alternatively, NPC2 may not be cholesterol-associated when secreted from astrocytes or the [3H]cholesterol radiolabel might have been too poorly represented in the pool from which NPC2 took its cholesterol.

The most intriguing finding in the present study was the abundance of the late intermediates of cholesterol biosynthesis found in astrocytes and in astrocyte-derived medium. The inefficient completion of cholesterol biosynthesis as detected by using radiolabeled acetate was reflected in the high percentage of unlabeled desmosterol in the cells as well as latho- and desmosterol in the medium as compared with cholesterol. Desmosterol has been previously reported in developing brain. In rat brain, desmosterol accumulates just before and during the early stages of myelination (37). Moreover, the amount of desmosterol was reported to increase during the first 2 weeks of life and, thereafter, rapidly decrease (38). Due to this timing, the high desmosterol level has been linked to the increased cholesterol biosynthesis taking place during myelination (37). Our study clearly demonstrates that high levels of desmosterol are not restricted to oligodendrocytes but are also a characteristic of embryonic astrocytes.

Cholesterol biosynthesis can occur either via lathosterol, which after conversion to 7-dehydrocholesterol is converted to cholesterol or, alternatively, via desmosterol to cholesterol (39). Age-related changes in the usage of these pathways have been reported. For instance, in amyloid precursor protein transgenic mouse brain, cholesterol was mainly synthesized by the desmosterol pathway postnatally, whereas in aged mice, lathosterol was the major precursor (40). The physiological significance of such variation remains open. Partial interchangeability of cholesterol and desmosterol at the whole animal level was recently demonstrated by the generation of desmosterol reductase-deficient mice (41). In these mice, desmosterol accounted for 99% of total sterols, yet the animals had a relatively mild phenotype.

In addition to astrocytes, desmosterol represents a major membrane sterol in sperm cells. Importantly, these cells have a very high percentage of polyunsaturated fatty acids in the membrane lipids (42), a characteristic that has been observed also in astrocytes (43). Moreover, desmosterol and docosahexanoic acid were unevenly distributed in the heads and tails of monkey sperm (44). It is conceivable that the ample amount of polyunsaturated fatty acids and desmosterol are linked together in the maintenance of optimal membrane structure both in sperm cells and in astrocytes. Such unusual membrane composition would be predicted to have implications for the biophysical behavior of the membrane.

An imminent question raised by this study is the functional role(s) of the sterol precursors in astrocytes and in astrocyte secretions. In the case of fibroblastic and hepatic cells, we have speculated that the precursor sterols released to extracellular acceptors might serve yet unknown signaling functions (12). A similar scenario could be extended to the secretory sterols in astrocytes. In addition, a structural or regulatory role in fine-tuning specific inter-lipid or lipid-protein interactions could be envisaged. Desmosterol is known to be a more potent regulator than cholesterol in down-regulating hydroxy-methyl-glutaryl-CoA reductase activity in glial cells (24). In addition, desmosterol is at least as efficient as cholesterol in modulating the conformation of the sterol-sensing domain protein SCAP (45). An important avenue for future work is to elucidate whether these close structural analogues of cholesterol play a role in the communication between astrocytes and neurons.


   FOOTNOTES
 
* This study was supported by Academy of Finland Grants 48905 and 49987, the Ara Parseghian Medical Research Foundation, the Sigrid Juselius Foundation, and the Helsinki Biomedical Graduate School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 358-9-191-59423; Fax: 358-9-191-59366; E-mail: elina.ikonen{at}helsinki.fi.

1 The abbreviations used are: NPC, Niemann-Pick type C disease; HPLC, high performance liquid chromatography; SFV, Semliki Forest virus; WT, wild type; PBS, phosphate-buffered saline. Back

2 A. Mutka and E. Ikonen, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Anna Uro, Liisa Arala, Birgitta Rantala, and Jari Metso for skillful technical assistance and Peter Lobel (Robert Wood Johnson Medical School) for kindly providing tissues from NPC2–/–, NPC2–/+, and NPC2+/+ mice.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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