MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein

Abstract

Niemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder, caused in most cases by mutations in the late endosomal protein NPC1. A hallmark of NPC disease is endosomal cholesterol accumulation and an impaired cholesterol homeostatic response, which might affect cholesterol transport to mitochondria and, thus, mitochondrial and cellular function. This study aimed to characterize mitochondrial cholesterol homeostasis in NPC disease. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to assess mitochondrial cholesterol import by pregnenolone production, we show that cholesterol transport to the mitochondrial inner membrane is not affected by loss of NPC1. However, mitochondrial cholesterol content was higher in NPC1-deficient than in wild-type cells. Cholesterol transport to the mitochondrial inner membrane increased markedly upon exposure of cholesterol-deprived cells to lipoproteins, indicating transport of endosomal cholesterol to mitochondria. Reduction of endosomal metastatic lymph node protein 64 (MLN64) by RNA interference decreased cholesterol transport to the mitochondrial inner membrane and reduced mitochondrial cholesterol levels in NPC1-deficient cells, suggesting that MLN64 transported cholesterol to mitochondria even in the absence of NPC1. In summary, this study describes a transport pathway for endosomal cholesterol to mitochondria that requires MLN64, but not NPC1, and that may be responsible for increased mitochondrial cholesterol in NPC disease.

Fig. 1.

Expression of F2-fusion protein of CYP11A1 complex confers the ability to convert cholesterol to pregnenolone in mitochondria. A, B: Localization of the F2 fusion protein to mitochondria. CHO cells transiently transfected with F2 were fixed and immunostained with antiferredoxin reductase antibodies (A) following incubation with Mitotracker Red (B). Images are representative of three independent experiments. Bars = 20 µm. C: CYP11A1 enzymatic activity was determined in crude mitochondria (mitochondria) and in the remaining cell fraction (supernatant) from CHO and F2-CHO cells (clone F2-CHO #6) as pregnenolone production from 22-OH-Chol in the presence of detergent and an NADPH regenerating system. Data represent means ± SEM of quadruplicate measurements.

Fig. 2.

F2-CHO and F2-4-4-19 cells grown in the presence of serum transport the same amount of cholesterol to the mitochondrial inner membrane. A: F2-complex and cyclophilin mRNA in four monoclonal CHO cell lines stably expressing F2 and in parental CHO cells measured by RT-PCR, with each cell line in triplicate (clone #8 in duplicate). Lane 1, 1 kB ladder; lane 16, 100 bp ladder. B: CYP11A1 enzymatic activity in cell lysates of the four F2 clones shown as pregnenolone production from 22-OH-Chol in the presence of detergent and an NADPH regenerating system. Data are means ± SEM of four independent experiments in duplicate. C: Representative CHO, F2-CHO, 4-4-19, and F2-4-4-19 cells were fixed and stained with filipin to visualize unesterified cholesterol. Bars = 40 µm. D: Pregnenolone formation from endogenous cholesterol sources during 6 or 24 h in intact, adherent F2-CHO and F2-4-4-19 cells. E: Maximum rate of pregnenolone formation from excess membrane-permeable precursor 22-OH-Chol during 24 h in intact, adherent F2-CHO and F2-4-4-19 cells. In D and E, data are means ± SEM, three independent experiments in triplicate.

Fig. 3.

Levels of cholesterol transported to the inner mitochondrial membrane are unchanged by siRNA-mediated depletion of NPC1. A: NPC1 protein levels in F2-CHO cells transfected with nontargeting siRNA (siNT) or siRNA directed against NPC1 (siNPC1), analyzed by immunoblotting. Tubulin was used as a loading control. Shown is one representative blot of five independent experiments. B, C: F2-CHO cells transfected with nontargeting siRNA (siNT; B) or siRNA directed against NPC1 (siNPC1; C) were stained with filipin to visualize cholesterol accumulation. Bars = 40 µm. D: Pregnenolone formed during 24 h from endogenous cholesterol sources in intact, adherent F2-CHO cells grown in the presence of serum and transfected with nontargeting siRNA (siNT) or siRNA directed against NPC1 (siNPC1) was measured as ng pregnenolone per mg cell protein and is depicted as percentage of the average of siNT. Mean ± SEM, five independent experiments [total n = 13 (siNT) or 16 (siNPC1)].

Fig. 4.

Endosomal and endogenously synthesized cholesterol is transported into mitochondria. A, C: Pregnenolone formation in intact, adherent F2-CHO (A) or F2-4-4-19 (C) cells grown in medium containing 5% LPDS and 50 µg/ml LDL for 48 h, then incubated in the presence (LDL→LDL) or absence (LDL→SF) of LDL in serum-free (SF) import medium for 24 h, or grown in medium containing 5% LPDS without LDL for 48 h, then incubated in the presence (LPDS→LDL) or absence (LPDS→SF) of LDL in serum-free import medium for 24 h. Pregnenolone was determined in import medium as ng pregnenolone per mg cell protein and is depicted as percentage of the average of the LDL→LDL incubation. B, D: Cholesterol biosynthesis in F2-CHO (B) or F2-4-4-19 (D) cells grown under the same conditions as in A and C with the addition of 1 µCi/ml [¹⁴C]acetate to the import medium. Cellular lipid extracts were separated by thin-layer chromatography, and radioactivity was counted in the band corresponding to unesterified cholesterol. Data are expressed as dpm per mg cell protein. All data are means ± SEM of three independent experiment in triplicate with two clones each F2-CHO and F2-4-4-19 cells (n = 18). * P < 0.05.

Fig. 5.

siRNA-mediated downregulation of MLN64 expression decreases cholesterol transport to the mitochondrial inner membrane. F2-CHO cells were transfected with nontargeting (siNT) siRNA, siRNA directed against MLN64 (siMLN64 #1 or siMLN64#3), or siMLN64#3 combined with siRNA against NPC1 (siMLN64/siNPC1) as indicated. A: Cell lysates analyzed by immunoblotting with antibodies directed against MLN64 and NPC1. Actin was used as loading control. B: Cells were grown in serum-containing medium; pregnenolone formation was measured in intact, adherent cells during 24 h in serum-free import medium. C: Cells were cholesterol deprived in medium with LPDS for 48 h; pregnenolone formation was measured in intact, adherent cells during 24 h in import medium containing LDL. Data are expressed as percentage of the average of siNT (measured as ng pregnenolone per mg cell protein), means ± SEM, four independent experiment with duplicate or triplicate measurements. * P < 0.05 compared with siNT.

Fig. 6.

Increased mitochondrial cholesterol content in 4-4-19 cells. A, B: Mitochondria were isolated from CHO and 4-4-19 cells grown in serum-containing medium by Percoll density ultracentrifugation or from wild-type and Npc1^−/− murine brain by immunoisolation as described in Materials and Methods. A: Unesterified cholesterol in mitochondria determined with the Amplex Red assay. B: Whole-cell homogenate (cells), crude mitochondria (crude mito), and pure mitochondria (pure mito) analyzed by immunoblotting with antibodies against VDAC (mitochondria), protein disulphide isomerase (endoplasmic reticulum), and NPC1 (late endosomes/lysosomes). C–F: Mitochondria were isolated from CHO and 4-4-19 cells grown in serum-containing medium by Percoll density ultracentrifugation after mild trypsinization. Where indicated, CHO cells were treated with 40 µM imipramine for 48 h prior to analysis. C: Electron micrograph of nontrypsinized and trypsinized mitochondria, 40,000× magnification. Bars = 500 nm. D: Immunoblot of crude mitochondria, and the mitochondrial and endosome-enriched fractions isolated by Percoll density ultracentrifugation following brief incubation with or without trypsin as described in Materials and Methods (pure mitochondria and endosomes). Fractions were prepared from CHO and 4-4-19 cells. Antibodies used as indicated. E: Unesterified cholesterol in pure mitochondria isolated from CHO, 4-4-19, or CHO cells treated with imip­ramine (imi), determined with the Amplex Red assay. Cholesterol data are standardized to mitochondrial protein and represent means ± SEM of three independent experiments (* P < 0.05). Immunoblots are representative of three independent experiments.

Fig. 7.

Mitochondrial cholesterol content is decreased following downregulation of MLN64 in 4-4-19 cells. Mitochondria were isolated from CHO and 4-4-19 cells transfected with nontargeting siRNA (siNT) or siRNA directed against MLN64 (siMLN64) and grown in serum-containing medium for 3 days. A: Immunoblot of cell lysates using anti-MLN64 antibodies. Actin was used as loading control on the same membrane. Shown is one immunoblot representative of three independent experiments. B, C: Unesterified cholesterol determined with the Amplex Red assay in mitochondria (B) and lysosomes/endosomes (C) isolated by Percoll gradient centrifugation. Data are standardized to mitochondrial protein and represent the means ± SEM of three independent experiments. * P < 0.05.