MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

Abstract

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Graphical abstract

Fig. 1

MLN64 overexpression increases mitochondrial cholesterol content in mice liver (A) MLN64 immunoblot. Total liver extracts (50 μg protein/lane) from Ad.E1Δ and Ad.MLN64 mouse livers 48 h after infection, run on 12% SDS-PAGE, transferred to PVDF membranes. (B) Mitochondria were isolated from Ad.E1Δ and Ad.MLN64 mouse livers 48 h after infection by Percoll density ultracentrifugation. The purity of mitochondria was confirmed by determining the distribution of specific markers such as CoxIV (mitochondria), Lamp1 and cathepsin B (lysosome) using immunoblot analysis. (C) Mitochondrial cholesterol content was quantified in the pure mitochondria fractions using HPLC. * Indicates statistically significant differences (p<0.05). (n Ad.E1Δ=5; n Ad.MLN64=5).

Fig. 2

MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

MLN64 overexpression produces mitochondrial dysfunction in hepatocytes (A) 48 h after Ad.E1Δ and Ad.MLN64 infection HepG2 cells were stained with MitoSOX Red to measure superoxide production and with Mitotracker Green to stain mitochondria respectively. Scale bar: 20 µm. (B) The graph shows the fluorescence intensity of MitoSOX as indicative of superoxide production. * p<0.05 (n=5). (C) MLN64 (green) and Mitotracker Red (red) staining of HepG2 cells 48 h after adenoviral infection. MMP levels were determined with Mitotracker red (MTred) staining and confocal imaging. MLN64 immunofluorescence was performed after cells were stained with MTred. The graph shows the quantification of MTred fluorescence intensity. * p<0.05 (n=5). Scale bar: 20 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

MLN64 overexpression triggers mitochondrial fragmentation in MEF and HepG2 cells. (A) MEF cells were infected with Ad.E1Δ and Ad.MLN64. Cerulean fluorescent protein in the outer mitochondrial membrane was analyzed 48 h after infection. Scale bar: 10 µm, zoom 5 µm. (B) The sphericity index was calculated from analysis of mitochondrial tridimensional morphology using the IMARIS software, (n=3). (C) Western blot analysis of mitochondrial dynamics proteins of HepG2 cells 48 h after adenoviral infection. OPA-1 and MFN-2 are mitochondrial fusion related proteins. FIS1 is a mitochondrial fission related protein. These proteins were analyzed by western blot and the bands intensity was normalized using the structural protein actin. * Indicates statistically significant differences (p<0.05) (n=3).

Fig. 5

Npc1^-/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p<0.05).

Fig. 6

NPC cells show mitochondrial morphological alterations and MLN64 down-regulation improves mitochondrial function in NPC cells. CHO Wild-type (WT) and Npc1^-/- (NPC) cells were transfected with nontargeting siRNA (siNT) and a siRNA directed against MLN64 (siMLN64) for 96 h. (A) 30 μg of proteins from cell homogenates were subjected to SDS-PAGE and analyzed by western blot using antibodies against the fission (FIS1) and fusion (MFN-2, OPA-1) related proteins . (B) The FIS1 bands intensities were measured using ImageJ and normalized with the ε-Cop protein. *p<0.05 (n=3) (C) Cells were immunostained with anti-MLN64 antibody (red), mitotracker green and nuclei were stained with Hoechst. (D) Cells were stained using Mitotracker red to measure the MMP or Mitosox (E) to quantify mitochondrial superoxide production 96 h post transfection. ^aStatistically different from WT. ^bStatistically different from NPC. ^cStatistically different from NPC+siNT. (n=3). Scale bar: 10 µm, zoom 5 µm.