Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells

Abstract

A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL. Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-beta-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2.

Fig. 1.

Role of NPC1 in sterol-mediated cholesteryl ester formation (A–C) and inhibition of SREBP-2 processing (D and E) in control and NPC1 mutant human fibroblasts. On day 0, nontransformed fibroblasts were set up in medium A with 10% FCS at 2 × 10⁴ cells per 60-mm dish. On day 3, cells were refed with the same medium, and on day 5, cells were switched to medium A with 10% human lipoprotein-deficient serum (LPDS). (A–C) Cholesterol esterification. On day 7, each dish received medium A containing 10% LPDS, 50 μM compactin, 50 μM sodium mevalonate, and the indicated concentration of LDL (A), 25-hydroxycholesterol (25-HC) (B), or cholesterol/methyl-β-cyclodextrin (MCD) complex (C). After 5 h at 37 °C, each monolayer was pulse-labeled for 2 h with 0.2 mM sodium [¹⁴C]oleate-albumin (7,966 dpm/nmol). The cells were harvested for measurement of the content of cholesteryl [¹⁴C]oleate. Each value is the average of duplicate incubations. (D and E) Immunoblot analysis of SREBP-2 processing. On day 7, after incubation for 5 h with the indicated concentration of LDL, 25-HC, or cholesterol/MCD, N-acetyl-leucinal-leucinal-norleucinal was added at a final concentration of 25 μg/mL. After 1 h at 37 °C, six dishes were harvested and pooled for preparation of nuclear extract and 100,000 × g membrane fractions, which were analyzed by immunoblotting as described in SI Materials and Methods. P, precursor form of SREBP-2; N, cleaved nuclear form of SREBP-2.

Fig. 2.

25-Hydroxycholesterol (25-HC) stimulated esterification of total cellular cholesterol in macrophages from wild-type and NPC1 mutant mice. On day 0, peritoneal macrophages from wild-type (A) or npc^nih/nih mice (B) were set up in medium B with 10% FCS as described in SI Materials and Methods. On day 1, the cells were incubated with medium B containing 10% FCS and 8.3 nM [³H]cholesterol (132,000 dpm/pmol; added in ethanol at a final concentration of 0.05%). After 24 h at 37 °C, each dish was washed with 5 mL of PBS with 0.2% BSA, after which the cells received fresh medium B with 0.2% BSA. After 12 h at 37 °C, each monolayer received medium B with 0.2% BSA in the absence or presence of 5 μg/mL 25-HC. After incubation for the indicated time, the cells were harvested for measurement of the content of [³H]cholesteryl ester (CE) and [³H]cholesterol, as described in SI Materials and Methods. Each value is the average of duplicate incubations and represents the percentage of [³H]cholesterol incorporated into [³H]CE relative to [³H]cholesterol content at zero time. Zero time values for [³H]cholesterol were 10.0 and 14.5 pmol/mg of protein for wild-type and npc^nih/nih macrophages, respectively.

Fig. 3.

25-Hydroxycholesterol (25-HC) stimulated esterification of newly synthesized cholesterol in fibroblasts from wild-type and NPC1 mutant mice. On day 0, fibroblasts from wild-type (A) and npc^nih/nih (B) mice were set up in medium B with 10% FCS and 4 × 10⁴ cells per 60-mm dish. On day 3, cells were switched to medium B with 5% newborn calf lipoprotein-deficient serum (LPDS). On day 4, cells were incubated in medium C with 5% LPDS and pulsed-labeled with 167 μM [³H]acetate (333 dpm/pmol) for 3 h at 14 °C to label newly synthesized cholesterol. After the pulse, each monolayer was washed twice with 3 mL of PBS with 0.2% BSA and then chased at 37 °C with medium B containing 5% LPDS, 50 μM compactin (to inhibit cholesterol synthesis), 50 μM sodium mevalonate, and 0.1 mM sodium oleate-albumin in the absence or presence of 3 μg/mL 25-HC. At the indicated time of chase, the cells were harvested for measurement of [³H]cholesteryl ester (CE) and [³H]cholesterol. Each value is the average of duplicate incubations and represents the percentage of [³H]cholesterol incorporated into [³H]CE relative to [³H]cholesterol content at zero time. Zero time values for cellular [³H]cholesterol were 160 and 192 pmol/mg of protein in wild-type and npc^nih/nih fibroblasts, respectively.

Fig. 4.

Inhibition of SREBP-2 processing by LDL-derived 25-hydroxycholesterol (25-HC), but not LDL-derived cholesterol, in NPC1 mutant human fibroblasts. On day 0, hTERT-control and hTERT-NPC1 human fibroblasts were set up in medium A with 10% FCS and 7 × 10⁴ cells per 60-mm dish. On day 2, the medium was switched to medium A with 5% human lipoprotein-deficient serum (LPDS). On day 4, the cells were incubated for 5 h in medium A containing 5% LPDS, 50 μM compactin, 50 μM sodium mevalonate, and one of the following additions: 1 μg/mL 25-HC (lanes B, J), 100 μg of protein per milliliter of LDL (lanes C, K), or the indicated concentration of LDL reconstituted with 25-HC oleate (r-[25-HC oleate]LDL) (lanes D–H, L–P). Cells then received N-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 μg/mL. After a further 1 h at 37 °C, triplicate dishes were harvested and pooled for preparation of nuclear extract and 100,000 × g membrane fractions, which were analyzed by immunoblotting for SREBP-2 and Scap as described in SI Materials and Methods. P, precursor form of SREBP-2; N, cleaved nuclear form of SREBP-2.

Fig. 5.

Cholesterol content of purified endoplasmic reticulum (ER) membranes from control and NPC1 mutant human fibroblasts. (A) Endoplasmic reticulum membrane fractionation scheme. (B and C) Immunoblot and enzymatic analysis of membrane fractions. On day 0, 13 dishes of hTERT-control and hTERT-NPC1 fibroblasts were set up in medium A with 10% FCS and 4 × 10⁵ cells per 100-mm dish. On day 3, the cells were harvested, and the ER membranes were prepared as described in SI Materials and Methods and shown in A. Equal volumes of each membrane fraction were subjected to immunoblot analysis for the indicated organelle markers (B) or assayed for the indicated enzyme activity (C). (D and E) Analysis of SREBP-2 cleavage (D) and cholesterol content of purified ER membranes (E). hTERT-control and hTERT-NPC1 fibroblasts (13 dishes for each condition) were set up as described above. On day 2, cells were switched to medium A with 5% human lipoprotein-deficient serum (LPDS). On day 3, cells were switched to medium A with 5% LPDS, 5 μM compactin, and 50 μM sodium mevalonate. On day 4, the cells received the same medium with 50 μM compactin and supplemented with either 100 μg of protein per milliliter of LDL or 20 μg/mL cholesterol/methyl-β-cyclodextrin (MCD) complex as indicated. After incubation for 6 h at 37 °C, the cells were harvested. (D) A portion of the harvested cells (5% of total) was fractionated and subjected to immunoblot analysis for SREBP-2. P, precursor form of SREBP-2; N, cleaved nuclear form of SREBP-2. (E) The remaining 95% of the harvested cells were used to purify the ER membranes. Lipids were extracted, and the amount of cholesterol and phospholipids was quantified as described in ref. . Cholesterol content in E is expressed as the molar percentage of total lipids (phospholipids + cholesterol). Each bar represents the mean ± SEM from five experiments.

Fig. 6.

Sphingomyelinase (SMase) treatment of control and NPC1 mutant fibroblasts stimulates cholesteryl ester (CE) synthesis. On day 0, hTERT-control and hTERT-NPC1 human fibroblasts were set up in medium A with 10% FCS at either 7 × 10⁴ cells per 60-mm dish (A and B) or 4 × 10⁵ cells per 100-mm dish (C). On day 2, cells were switched to medium A with 5% human lipoprotein-deficient serum (LPDS). (A) Immunoblot analysis of SREBP-2 cleavage. On day 4, the cells were incubated with medium A containing 5% LPDS, 50 μM compactin, 50 μM sodium mevalonate, 0.1% DMSO, and 10 μg/mL ACAT inhibitor Sandoz 58-035. After 30 min at 37 °C, cells received SMase (200 milliunits per milliliter), β-migrating very low density lipoprotein (β-VLDL) (10 μg of protein per milliliter), or 25-hydroxycholesterol (25-HC) (1 μg/mL) as indicated. After 2 h at 37 °C, triplicate dishes were harvested and pooled for preparation of nuclear extract and 100,000 × g membrane fractions, which were analyzed by immunoblotting for SREBP-2. P, precursor form of SREBP-2; N, cleaved nuclear form of SREBP-2. (B) Cholesterol esterification assay. On day 4, each dish received medium A with 5% LPDS, 50 μM compactin, and 50 μM sodium mevalonate in the absence or presence of 200 milliunits per milliliter of SMase. After 1 h at 37 °C, each monolayer was pulse-labeled for 1 h with 0.2 mM sodium [¹⁴C]oleate-albumin (32,433 dpm/nmol). Cells were harvested for measurement of cholesteryl [¹⁴C]oleate. Each value is the average of duplicate incubations. (C) Analysis of the cholesterol content of purified endoplasmic reticulum (ER) membranes. On day 3, cells from 13 dishes (100 mm) for each condition were switched to medium A containing 5% LPDS, 5 μM compactin, and 50 μM sodium mevalonate. On day 4, cells received the same medium supplemented with 50 μM rather than 5 μM compactin in the absence or presence of 200 milliunits per milliliter of SMase. After 2 h at 37 °C, the cells were harvested, and the ER membranes were purified as described in SI Materials and Methods and Fig. 5. Cholesterol and phospholipids were quantified as described in Fig. 5E. Cholesterol content is expressed as the molar percentage of total lipids (phospholipids + cholesterol). Each bar represents the mean ± SEM from three experiments.

Fig. 7.

Cholesteryl ester formation in control, NPC1 mutant, and NPC2 mutant human fibroblasts treated with 2-hydroxypropyl-β-cyclodextrin (HPCD). (A–D) On day 0, nontransformed control fibroblasts, NPC1 mutant fibroblasts, and NPC2 mutant fibroblasts were set up in medium A with 10% FCS and 2 × 10⁴, 2 × 10⁴, and 7 × 10⁴ cells per 60-mm dish, respectively. On day 3, cells were refed with the same medium. (A and B) On day 5, cells were switched to medium A with 10% human lipoprotein-deficient serum (LPDS). On day 7, each dish received medium A containing 10% LPDS, 50 μM compactin, and 50 μM sodium mevalonate in the absence or presence of 100 μg of protein per milliliter of LDL (A) or 5 μg/mL 25-hydroxycholesterol (25-HC) (B). After 5 h at 37 °C, each monolayer was pulse-labeled for 2 h with 0.2 mM sodium [¹⁴C]oleate-albumin (8,187 dpm/nmol). The cells then were harvested for measurement of cholesteryl [¹⁴C]oleate. (C and D) On day 6, cells were incubated with medium A with 10% FCS and 20 μg of protein per milliliter of LDL. After 16 h, each dish received medium A containing 10% FCS and 0.1% (wt/vol) HPCD (C) or the indicated concentration of HPCD (D). After incubation for the indicated time in C or after 6 h in D, each monolayer was pulse-labeled for 2 h with 0.2 mM sodium [¹⁴C]oleate-albumin (8,187 dpm/nmol). The cells then were harvested for measurement of cholesteryl [¹⁴C]oleate. (A–D) Each value is the average of duplicate incubations.