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Human Molecular Genetics Advance Access originally published online on April 27, 2007
Human Molecular Genetics 2007 16(12):1495-1503; doi:10.1093/hmg/ddm100
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Autophagy in Niemann–Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects

Chris D. Pacheco1, Robin Kunkel2 and Andrew P. Lieberman1,2,*

1 Neuroscience Program and 2 Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA

* To whom correspondence should be addressed at: Department of Pathology University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA. Tel: +1 7346474624; Fax: +1 7346153441; Email: liebermn{at}umich.edu

Received February 7, 2007; Revised April 6, 2007; Accepted April 11, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Niemann–Pick C (NPC) disease is an autosomal recessive lipid storage disorder characterized by a disruption of sphingolipid and cholesterol trafficking that produces cognitive impairment, ataxia and death, often in childhood. Most cases are caused by loss of function mutations in the Npc1 gene, which encodes a protein that localizes to late endosomes and functions in lipid sorting and vesicle trafficking. Here, we demonstrate that NPC1-deficient primary human fibroblasts, like npc1–/– mice fibroblasts, showed increased autophagy as evidenced by elevated LC3-II levels, numerous autophagic vacuoles and enhanced degradation of long-lived proteins. Autophagy because of NPC1 deficiency was associated with increased expression of Beclin-1 rather than activation of the Akt-mTOR-p70 S6K signaling pathway, and siRNA knockdown of Beclin-1 decreased long-lived protein degradation. Induction of cholesterol trafficking defects in wild-type fibroblasts by treatment with U18666A increased Beclin-1 and LC3-II expression, whereas treatment of NPC1-deficient fibroblasts with sphingolipid-lowering compound NB-DGJ failed to alter the expression of either Beclin-1 or LC3-II. Primary fibroblasts from patients with two other sphingolipid storage diseases, NPC2 deficiency and Sandhoff disease, characterized by sphingolipid trafficking defects also showed elevation in Beclin-1 and LC3-II levels. In contrast, Gaucher disease fibroblasts, which traffic sphingolipids normally, showed wild-type levels of Beclin-1 and LC3-II. Our data define a critical role for Beclin-1 in the activation of autophagy because of NPC1 deficiency, and reveal an unexpected role for lipid trafficking in the regulation of this pathway in patients with several sphingolipid storage diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sphingolipid storage diseases encompass a group of ~40 genetically distinct disorders that result from inherited deficiencies of lysosomal hydrolytic activities or lipid transport. These disorders occur with a collective frequency of 1 in 8000 live births (1), and are often associated with devastating neurodegeneration. Among this group is Niemann–Pick C (NPC) disease, an autosomal recessive disorder of lipid trafficking that produces cognitive impairment, ataxia and death, most often in childhood (2). NPC disease is characterized by the accumulation of unesterified cholesterol and sphingolipids in late endosomes and lysosomes. Nine-five percent of NPC disease patients have loss of function mutations in the Npc1 gene (3). The encoded multipass transmembrane protein contains a sterol-sensing domain (4) and functions in late endosomes to promote lipid sorting and vesicular trafficking (5–8) through mechanisms that are incompletely understood.

Mice deficient in NPC1, which reproduce the pathology and lipid trafficking defects of NPC disease, arose from a spontaneous mutation in the Npc1 gene (npc1–/– mice) (9). Similar defects occur in chimeric mice that lack functional NPC1 in only some cells (10). In both cases, NPC1 deficiency leads to the activation of macroautophagy (hereafter referred to as autophagy) in the cerebellum, a process by which cytoplasmic proteins and organelles are sequestered within autophagosomes and are targeted for degradation by lysosomes (11). This regulated and evolutionarily conserved pathway enables recycling of limited or damaged cellular constituents to promote cell survival. However, in other instances, robust activation of autophagy leads to cell death.

Here, we have used npc1–/– mice and primary human fibroblasts deficient in NPC1 to explore the mechanism by which autophagy is induced in NPC disease. Our data demonstrate that enhanced basal autophagy in NPC1 deficiency is mediated by increased expression of Beclin-1 rather than by activation of the Akt-mTOR-p70 S6K pathway. We further demonstrate that lipid trafficking defects caused by pharmacologic treatment or by human disease gene mutations occurring in other sphingolipid storage diseases also up-regulate Beclin-1 and result in an increased autophagy. Our findings establish Beclin-1 as a critical regulator of autophagy in several sphingolipid storage diseases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NPC1 deficiency increases basal autophagy
We first sought to determine whether elevated levels of autophagy occur specifically in the central nervous system of NPC1-deficient (npc1–/–) mice or also occur in other organs that exhibit pathology. To accomplish this, we used the microtubule-associated protein 1 light chain 3 (LC3) as a marker of autophagy. This protein is modified from its LC3-I cytosolic form to a more rapidly migrating, lipid-conjugated LC3-II form associated with autophagosome membranes when autophagy is induced (12,13). Cerebellar and liver lysates from 6-week-old npc1–/– mice had elevated levels of LC3-II compared with wild-type littermates (Fig. 1A), demonstrating that NPC1 deficiency increased autophagy in both organs. Similarly, primary human fibroblasts deficient in NPC1 expressed higher total LC3 and LC3-II levels than control fibroblasts (Fig. 1B). This difference was observed in untreated cells and following starvation or rapamycin treatment, indicating that basal levels of autophagy were increased by NPC1 deficiency and that pathways leading to its further activation were intact in mutant cells.


Figure 1
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  		Figure 1. Increased autophagy in NPC1-deficient mice and human fibroblasts. (A) Cerebellar (lanes 1 and 2) and liver (lanes 3 and 4) lysates from 6-week-old wild-type (lanes 1 and 3) and npc1–/– (lanes 2 and 4) mice were examined by western blot for expression of LC3 (top) and ß-tubulin (bottom). (B) Lysates from control (lanes 1–3) and NPC1-deficient human fibroblasts (lanes 4–6) were collected from untreated cells (lanes 1 and 4), following 2 h starvation (lanes 2 and 5) or 24 h rapamycin (1 µM) treatment (lanes 3, 6). LC3 (top) and ß-tubulin (bottom) were visualized by western blot. (C) Electron micrographs of control human fibroblast (left panel) with infrequent cytoplasmic vacuoles, and NPC1-deficient fibroblasts (middle and right panels) with frequent vacuoles. Middle panel shows low and high (inset) magnification images of membrane-bound vacuoles with cytoplasmic contents consistent with autophagosomes or autolysosomes. Right panel shows autophagic vacuoles and mutilamellar bodies. Scale bars in lower right. N is nucleus. (D) MDC staining of control and NPC1-deficient human fibroblasts normalized to cell number (mean±SD). P < 0.02 by unpaired Student's t-test. (E) Lysates from NPC1-deficient human fibroblasts stably expressing FLAG-tagged NPC1 protein (lane 2) or empty vector (lane 1) were examined by western blot for expression of NPC1 (anti-FLAG, top), LC3 (middle) and ß-tubulin (bottom).

 
High levels of basal autophagy in NPC1-deficient fibroblasts were confirmed by transmission electron microscopy (Fig. 1C). This analysis demonstrated frequent autophagic vacuoles containing rough ER and other cytoplasmic contents in mutant, but not in wild-type fibroblasts. Similarly, staining with monodansylcadaverine (MDC), a dye that preferentially incorporates into autophagic vacuoles (14,15), was significantly higher in NPC1-deficient fibroblasts than that in control fibroblasts (Fig. 1D). To determine whether exogenous NPC1 could decrease autophagy in mutant fibroblasts, we stably expressed NPC1 protein in null cells. We observed decreased total LC3 and LC3-II levels in pooled, NPC1- transfected cells (Fig. 1E), demonstrating diminished basal autophagy levels.

We next sought to determine whether the autophagic pathway was intact in NPC1-deficient cells. LC3-II is degraded following fusion of autophagosomes with lysosomes (16). Failure to complete this step, as seen in other lysosomal storage diseases (17), increases LC3-II levels yet renders them resistant to further elevation upon inhibition of lysosomal proteases (16). Treatment of both wild-type and NPC1-deficient fibroblasts with lysosomal protease inhibitors E64d and pepstatin A similarly increased LC3-II levels, consistent with the notion that fusion of autophagosomes to lysosomes was intact in mutant cells (Fig. 2A). This conclusion was supported by the additive effect of concurrent induction of autophagy by rapamycin and inhibition of lysosomal proteases by E64d and pepstatin A.


Figure 2
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  		Figure 2. The autophagic pathway is intact in NPC1 deficiency. (A) Lysates from control (lanes 1–4) and NPC1-deficient (lanes 5–8) human fibroblasts were collected from untreated cells (lanes 1 and 5) or following 24 h treatment with rapamycin (1 µM) or E64d (10 µg/ml) with pepstatin A (10 µg/ml), as indicated. Expression of LC3 (top) and ß-tubulin (bottom) were determined by western blot. (B) Degradation of long-lived proteins in control (solid line) and NPC1-deficient (dashed line) human fibroblasts. Data (mean±SEM) are reported relative to control cells at 24 h. P = 0.0006 at 6 h, 0.01 at 18 h and 0.005 at 24 h by unpaired Student's t-test.

 
As an independent confirmation that the autophagic pathway was intact in NPC1-deficient cells, we measured the degradation of long-lived proteins (18). This assay provides a functional readout since autophagy is the major pathway through which many of these proteins are degraded (19,20). Wild-type and NPC1-deficient fibroblasts were labeled with 3H–leucine for 48 h, then washed and re-fed, and trichloroacetic acid (TCA) soluble radioactive counts were measured in the medium after 6, 18 and 24 h (Fig. 2B). Significantly higher levels of proteolysis were detected by NPC1-deficient fibroblasts at all points, demonstrating increased protein turnover in cells that also exhibited enhanced autophagy.

Beclin-1 mediates increased levels of autophagy in NPC1 deficiency
The induction of autophagy is achieved through either the dephosphorylation of mTOR or the activation of the Beclin-1 pathway (11). To determine which of these was preferentially activated because of NPC1 deficiency, protein lysates from mutant mice and fibroblasts were examined by western blot (Fig. 3). Beclin-1, an evolutionarily conserved protein that is part of the Class III PI3K complex that participates in autophagosome formation (21), was expressed at mildly increased levels in both cerebellum and liver of npc1–/– mice compared with wild-type littermates (Fig. 3A). Increased Beclin-1 expression was also observed in NPC1-deficient fibroblasts (Fig. 3B), demonstrating that up-regulation of Beclin-1 occurred in response to NPC1 deficiency in mice and cells.


Figure 3
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  		Figure 3. NPC1 deficiency causes Beclin-1 up-regulation, but not mTOR activation. (A) Cerebellar (lanes 1 and 2) and liver (lanes 3 and 4) lysates from wild-type (lanes 1 and 3) and npc1–/– (lanes 2 and 4) mice were examined by western blot for expression of Beclin-1 (top) and GAPDH (bottom). (B) Lysates from untreated control (lane 1) and NPC1-deficient (lanes 2) human fibroblasts were analyzed by western blot for expression of Beclin-1 (top) and ß-tubulin (bottom). (C) Cerebellar (C) and liver (L) lysates from wild-type (lanes 1 and 2) and npc1–/– (lanes 3 and 4) mice were probed for phosphorylated-Akt, total Akt, phosphorylated-mTOR and total mTOR and visualized by western blot. (D) Lysates from control (lanes 1 and 2) and NPC1-deficient (lanes 3 and 4) fibroblasts were collected from untreated cells (lanes 1 and 3) or from cells following 2 h starvation (lanes 2 and 4; Akt rows) or 24 h rapamycin (1 µM) treatment (lanes 2 and 4; p70 S6K rows). Expression of phosphorylated-Akt (upper band = specific), total Akt, phosphorylated-mTOR, total mTOR, phosphorylated-p70 S6K and total p70 S6K were visualized by western blot.

 
In contrast, our analyses did not reveal activation of the mTOR pathway as a consequence of NPC1 deficiency. No alteration in the phosphorylation of mTOR, its regulator Akt or its target p70 S6K was detected in npc1–/– mice or NPC1-deficient fibroblasts (Fig. 3C and D). We did, however, observe stimulation of the mTOR pathway in NPC1-deficient fibroblasts as evidenced by starvation-induced Akt dephosphorylation or rapamycin-induced p70 S6K dephosphorylation, confirming that this pathway was intact in mutant cells. These data demonstrated that NPC1 deficiency did not activate the Akt-mTOR-p70 S6K signaling pathway in cell culture or in mice.

To determine the extent to which Beclin-1 up-regulation mediated enhanced basal autophagy in NPC1-deficient fibroblasts, we used pooled, targeted siRNAs to specifically knockdown Beclin-1 expression (Fig. 4A). Treatment with Beclin-1 siRNAs significantly decreased degradation of long-lived proteins in NPC1-deficient fibroblasts, but not in wild-type fibroblasts (Fig. 4B). We conclude that activation of Beclin-1, and not the mTOR pathway, mediated increased basal autophagy in NPC1-deficient fibroblasts.


Figure 4
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  		Figure 4. siRNA knockdown of Beclin-1 is sufficient to decrease basal autophagy in NPC1-deficient fibroblasts. (A) Lysates from mock (lane 1), non-targeted siRNA (lane 2) and Beclin-1 siRNA (lane 3) transfected NPC1- deficient fibroblasts were analyzed by western blot for expression of Beclin-1 (top) and ß-tubulin (bottom). (B) Degradation of long-lived proteins in control and NPC1-deficient human fibroblasts following transfection with non-targeted siRNA (black bars) or Beclin-1 siRNA (white bars). Data (mean±SEM) are reported relative non-targeted siRNA-transfected cells at 24 h. Relative proteolysis is significantly decreased in NPC1-deficient cells (P = 0.018 by unpaired Student's t-test) but not in controls (P = 0.435).

 
Defects in lipid trafficking lead to up-regulation of Beclin-1
NPC disease is characterized by the accumulation of both unesterified cholesterol and glycosphingolipids in late endosomes and lysosomes. This trafficking defect creates a functional deficit of cholesterol in other intracellular compartments (22–24). It was recently demonstrated that autophagy is responsive to intracellular cholesterol levels (25,26). We therefore treated control fibroblasts with U18666A, a compound known to induce an accumulation of unesterified cholesterol similar to that occurring in NPC1-deficient fibroblasts (Fig. 5A) (27). U18666A treatment of control fibroblasts resulted in increased expression of both LC3-II and Beclin-1 (Fig. 5B), suggesting that cholesterol trafficking defects were sufficient to activate autophagy and increase Beclin-1 expression.


Figure 5
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  		Figure 5. Unesterified cholesterol accumulation increases Beclin-1 and LC3-II expression. (A) Filipin staining of unesterified cholesterol in untreated control and NPC1-deficient fibroblasts, and control fibroblasts after 24 h treatment with U18666A (1 µg/ml). (B) Lysates from untreated (lane 1) and U18666A-treated (1 µg/ml, 24 h) control fibroblasts (lane 2) were analyzed for expression of LC3 (top), Beclin-1 (middle) and ß-tubulin (bottom) by western blot. (C) Lysates from untreated, NPC1-deficient fibroblasts (lane 1) or following treatment with NB-DGJ (50 µM) for 1, 3 or 5 days (lanes 2–4) were analyzed for expression of LC3 (top) and Beclin-1 (bottom) by western blot.

 
To evaluate the effect of glycosphingolipids on the induction of autophagy, NPC1-deficient cells were treated with the imino sugar N-butyl-deoxygalactonojirimycin (NB-DGJ). Although this compound decreases glycosphingolipid levels in NPC1-deficient cells (28), NB-DGJ treatment did not alter LC3-II or Beclin-1 expression (Fig. 5C). These findings support the notion that elevated basal autophagy in NPC1-deficient cells was primarily a consequence of altered lipid trafficking rather than glycosphingolipid accumulation.

To further explore the relationship between lipid trafficking defects, Beclin-1 expression and autophagy, we used a panel of primary human fibroblasts derived from patients with several sphingolipid storage diseases. NPC2 deficiency, which results in a clinical and biochemical phenocopy of NPC1 deficiency and causes ~5% of NPC disease (29), resulted in elevated basal levels of Beclin-1 and LC3-II (Fig. 6A and B). Sandhoff disease fibroblasts, which traffic sphingolipids abnormally (30–32), similarly exhibited elevated basal Beclin-1 and starvation-induced LC3-II levels (Fig. 6A and B). In contrast, Gaucher disease fibroblasts, which lack sphingolipid trafficking defects (30–32), showed wild-type basal levels of Beclin-1 and starvation-induced LC3-II expression (Fig. 6A and B). Taken together, these analyses revealed an unexpected contribution of lipid trafficking defects to the regulation of autophagy by altering the expression of Beclin-1.


Figure 6
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  		Figure 6. Other sphingolipidoses, which are characterized by lipid trafficking defects, display increased Beclin-1 expression. (A) Lysates from untreated control fibroblasts (lane 1), or from patients with NPC2 deficiency (lane 2), Gaucher disease (lane 3) and Sandhoff disease (lane 4) were analyzed for expression of Beclin-1 (top) and ß-tubulin (bottom) by western blot. (B) Lysates were collected from untreated control (lane 1), NPC1-deficient (lane 2) and NPC2-deficient (lane 3) fibroblasts, and from 2 h serum starved control (lane 4), Gaucher disease (lane 5), NPC1-deficient (lane 6) and Sandhoff disease (lane 7) fibroblasts. Expression of LC3 (top) and ß-tubulin (bottom) were determined by western blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The studies reported here help define the relationship among defects in lipid trafficking, autophagy and disease. Cholesterol and sphingolipid accumulations in late endosomes and lysosomes of NPC1-deficient cells are biochemical hallmarks of NPC disease. This disruption of lipid trafficking is associated with the induction of autophagy. Prompted by recent reports demonstrating increased autophagy following depletion of intracellular cholesterol (25,26), we explored the mechanism of autophagy up-regulation using primary human fibroblasts and mice deficient in NPC1. Our data establish that NPC1 deficiency leads to an increased basal autophagy as evidenced by elevated LC3-II levels, frequent autophagic vacuoles, increased MDC staining and enhanced degradation of long-lived proteins. This induction of autophagy is associated with increased expression of Beclin-1 rather than activation of the Akt-mTOR-p70 S6K pathway. Down- regulation of Beclin-1 by siRNA decreased the degradation of long-lived proteins in NPC1-deficient fibroblasts, but not wild-type fibroblasts, demonstrating a critical role for Beclin-1 in the regulation of basal autophagy rates.

Beclin-1 is the mammalian ortholog of yeast Atg6, and is a cytosolic protein that is part of the Class III PI3K machinery that participates in autophagosome formation (21). In addition to its role as a regulator of autophagy, Beclin-1 interacts with the anti-apoptotic protein Bcl-2, providing an intriguing link between pathways controlling autophagy and cell death (33). The interaction of Beclin-1 with Bcl-2 inhibits autophagy, while Beclin-1 expression in the absence of Bcl-2 binding potently induces autophagy (33). Thus, induction of Beclin-1 expression in NPC disease may simultaneously affect autophagy and cell survival. Interestingly, pharmacological disruption of cholesterol trafficking, which we show induces autophagy, also triggers apoptosis in cultured neurons (34,35). However, a clear role for apoptosis in NPC neuropathology has not yet been demonstrated (36) and autophagy may influence cell viability through other mechanisms.

Beclin-1 expression was increased by pharmacological treatments that cause an accumulation of unesterified cholesterol and by human disease gene mutations that disrupt lipid trafficking. Treatment of wild-type fibroblasts with U18666A and examination of primary cells from patients with NPC2 deficiency and Sandhoff disease revealed increased Beclin-1 levels, consistent with the notion that expression of this autophagy regulator is elevated in cells with defective cholesterol or sphingolipid transport. In contrast, Gaucher disease fibroblasts both traffic sphingolipids normally (30–32) and express wild-type levels of Beclin-1 and LC3-II. Although Beclin-1 levels were elevated in Sandhoff disease fibroblasts, basal LC3-II levels were not increased (data not shown) and expression beyond wild-type levels was detected only after serum starvation. We conclude that Beclin-1 up-regulation is necessary but not sufficient to increase basal autophagy. The enhanced response of Sandhoff disease fibroblasts to serum starvation suggests that elevated Beclin-1 levels prime cells for autophagy induction in response to additional signals.

Our observations indicate that the Beclin-1–Class III PI3K complex is a critical regulator of autophagy in several sphingolipid storage diseases. Activation of this pathway may promote cell survival by generating building blocks that are otherwise limiting, as is well characterized during periods of amino acid deprivation (11), or by facilitating the removal of damaged organelles or toxic proteins. We favor a model in which enhanced basal autophagy in NPC1-deficient cells acts in this manner to promote cell survival. This notion is consistent with recently characterized Atg5 and Atg7 null mice in which suppression of autophagy leads to neurodegeneration (37–39). Autophagy has also been implicated in non-apoptotic cell death, and its robust activation in starved or rapamycin treated cells may lead to cellular demise. This type of cytotoxicity has been demonstrated in bax/bak-deficient cells that undergo caspase-independent, autophagic cell death following etopiside treatment (40). Our data suggest that modulating activity of the Beclin-1–Class III PI3K complex and altering rates of autophagy may be a promising therapeutic approach for several sphingolipid storage diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Mouse samples were from age- and sex-matched BALB/cNctr-Npc1m1N/J (Jackson Laboratories stock number 003092) and wild-type littermates. Human fibroblasts from age- and sex-matched donors were from Coriell Cell Repositories (control cells, GM00038C; NPC1-deficient, GM03123A; NPC2-deficient, GM17910; Gaucher disease, GM10915 and Sandhoff disease, GM11707). Filipin, rapamycin, E64d, pepstatin A, anti-FLAG M2 monoclonal antibody, G418 and U18666A were from Sigma. NB-DGJ was from Toronto Research Chemicals. ß-Tubulin and Beclin-1 (BECN1) antibodies were from Santa Cruz Biotechnology. Phospho-Akt (Ser473), total Akt, phospho- p70 S6K (Thr412) and total p70 S6K antibodies were from Upstate. Phospho-mTOR (Ser2448) and total mTOR antibodies were from Cell Signaling. GAPDH antibody was from Abcam. Anti-LC3 antibody was a gift from Dr Tamotsu Yoshimori. Secondary HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were from BioRad.

Cell culture
Fibroblasts were maintained at 37°C, 5% CO2 in MEM with Earle's salts and non-essential amino acids (Gibco), supplemented with 15% FBS (Atlanta Biologicals) and 10 µg/ml penicillin, 10 µg/ml streptomycin and 2 mM glutamine (referred to as complete MEM medium).

Western blot analysis
Cells were harvested, washed with PBS and lysed in RIPA buffer containing cOmplete Protease Inhibitor Cocktail (Roche Diagnostics) and 0.1% ß-mercaptoethanol. Liver and cerebellar samples were homogenized with the same buffer using a motor homogenizer. Lysates were precleared by centrifugation at 15 000g for 10 min at 4°C. Samples were electrophoresed through either a 10% SDS–polyacrylamide gel or a 4–20% Tris–glycine gradient gel (Cambrex) and then transferred to Immunobilon-P (Millipore) or nitrocellulose membranes (BioRad) using a semidry transfer apparatus. Immunoreactive proteins were detected by chemiluminescence (PerkinElmer). Western blots depicted show representative results from one of three experiments.

Electron microscopy
Cells were fixed in suspension with 4% glutaraldehyde in 0.1 M calcodylate buffer, pH 7.3, overnight at 4°C, and then post-fixed for 1 h at room temperature in 2% osmium tetraoxide in 0.1 M cacodylate. After dehydration with graded ethanols and propylene oxide, cells were embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed on a Philips 400T transmission electron microscope.

MDC staining
Cells were seeded in six-well plates at equal density in complete MEM medium for 24 h and then stained with MDC as previously described (15). Briefly, cells were incubated with 0.05 mM MDC in PBS for 10 min, then harvested, washed with PBS and collected in 10 mM Tris–HCl, pH 8.0, with 0.1% Triton X-100. Fluorescence of incorporated intracellular MDC (excitation wavelength 390 nm, emission filter 527 nm) was measured by an Ascent Fluroskan microplate reader. To normalize for cell number, a final concentration of 0.2 µM ethidium bromide was added to the suspension and DNA fluorescence was measured (excitation wavelength 544 nm, emission filter 590 nm).

pCMV–mNPC1–3xFLAG expression construct and stable line
The mouse NPC1 cDNA was amplified from pBS-KS(+)mNPC1 (gift from Dr William Pavan) and an Asp718 site was added at the 3' end. An intermediate construct was generated by ligation of a 1 kb Asp718/EcoR1 fragment from the mNPC1 PCR product to the p3XFLAG-CMV-14 vector (Sigma) after Asp718/EcoR1 digestion. The final expression vector was generated by ligation of a 3 kb EcoRI fragment from the pBS-KS(+)mNPC1 plasmid to the EcoRI linearized intermediate construct. All amplified regions and cloning sites were verified by restriction digestion and sequencing.

The plasmid was electroporated into NPC1-deficient human fibroblasts with the Amaxa kit for normal human dermal fibroblasts (adult) using program U-23 on the Amaxa Nucleofector. Cells were plated in complete MEM medium for 2 days, and then selected with 1 mg/ml G418 in complete MEM medium for 1 week. Cells were allowed to recover from G418 selection for 3 days and were harvested for western blot.

Measurement of long-lived protein degradation
Degradation of long-lived proteins was determined using the published method (18) with minor modifications. Cells were seeded in 12-well plates in complete MEM for 24 h and allowed to grow to 70–80% confluency. After washing with HBSS, cells were labeled with 2 µCi/ml 3H-leucine (Amersham) in complete MEM medium. After 48 h of labeling, cells were washed with HBSS and incubated with complete MEM supplemented with 2.8 mM leucine. Aliquots of medium were collected at the indicated times, 20% TCA was added and samples were stored at 4°C. Upon acquisition of all samples, BSA (final concentration 3 mg/ml) was added, samples were incubated at 4°C for 1 h and then centrifuged at 15 000g for 5 min at 4°C. Supernatants were collected, the pellets were washed twice with cold 20% TCA, then all supernatants were pooled and the total radioactivity was measured by scintillation counting. After the last time point was collected, cells were washed with PBS and incubated for 1 h at 37°C in 0.1 N NaOH/0.1% Na deoxycolate. An aliquot of the solubilized cells was used to determine total protein concentration (BioRad). Relative proteolysis was determined by normalizing TCA soluble radioactivity in the medium to protein concentration from the solubilized cells.

siRNA knockdown of Beclin-1
Fibroblasts growing in complete MEM media were collected using the Reagent Pack subculture reagent kit (BioWhitaker), resuspended in Nucleofector solution (human dermal fibroblast Nucleofector kit, Amaxa) and mixed with 1.5 µg ON-TARGETplus SMART pool human BECN1 (NM_ 003766), siRNA (L-010552-00-0005) or siCONTROLplus non-targeting pool (D-001810-10-05) (Dharmacon). The suspension was electroporated using the program U-23 on the Amaxa Nucleofector. Cells were plated in 12-well dishes and, 4 h later, were used to measure protein degradation. Alternatively, cell lysates were collected 24 h after electroporation for western blot.

Filipin staining
Cells were seeded in chamber slides and incubated in complete MEM medium for 24 h. After washing with PBS, cells were fixed in 3% paraformaldehyde for 1 h at room temperature, washed with PBS and incubated with 1.5 mg/ml glycine in PBS for 10 min at room temperature. Cells were then stained for 2 h with 0.05 mg/ml filipin in PBS supplemented with 10% FBS at room temperature. Staining was seen by fluorescence microscopy using the UV filter set on a Zeiss Axioplan 2 imaging system.


   ACKNOWLEDGEMENTS
 
We thank Dr Tamotsu Yoshimori for the LC3 antibody, Dr William Pavan for the mouse NPC1 cDNA, Dr Daniel Klionsky for helpful comments, Dr Ana Maria Cuervo for the protein degradation assay protocol and Elizabeth Horn for the preparation of the figures. This work was supported by the National Institutes of Health through a Kirschstein NRSA predoctoral fellowship (NS51143) to C.D.P and a grant (HL 031963) to R.K.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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