Nt protects cells from LMP-dependent apoptosis [20]. Although this finding, which has also been confirmed by others [21], may seem counterintuitive, it is possibleLysosomal Stability Is Regulated by CholesterolFigure 2. Manipulation of lysosomal cholesterol content modulates the cellular sensitivity to apoptosis. Cholesterol content of human fibroblasts was AKT inhibitor 2 site modulated using U18666A, quinacrine, methyl-b-cyclodextrin (MbCD) or 25-hydroxy cholesterol (25-HC) before apoptosis was induced using O-methyl-serine dodecylamide hydrochloride (MSDH; 24 h). Phase contrast images of A) wt and B) NPC1-mutant fibroblasts (NPC1mut). Scale bar 20 mm. C) Viability of cultures in A and B, respectively, assessed by the MTT assay (n = 4). Viability is expressed as percentage of untreated cultures. D) Caspase-3 like activity (n = 4?). E) Representative curve of increase in green fluorescence during photo-oxidation of acridine orange. F) Quantification of lag time (as presented in E; n = 5?). Data are presented as the mean 6 SD, * p#0.05. doi:10.1371/journal.pone.0050262.gthat cholesterol preserves the integrity of the lysosomal membrane and thus promotes neuronal survival upon acute cellular stress. Importantly, in both NPC1-mutant cells and U18666A treated cells, cholesterol accumulation is associated with storage of severalother lipids [9,22], which might influence lysosomal stability. In addition, the expression of LAMP-2 was increased by U18666A treatment [20]. Because LAMPs have been shown to be important for regulation of LMP [23], Methionine enkephalin web further studies to distinguish betweenLysosomal Stability Is Regulated by Cholesterolthe LMP-modulating roles of cholesterol, sphingolipids and altered LAMP-1 and 22 expression were undertaken. We hypothesize that modulation of lysosomal composition affects cellular sensitivity to apoptosis and cell fate can be manipulated by the use of agents inducing or reducing cholesterol content. We herein provide evidence that cholesterol, and not accompanying sphingolipids or LAMP proteins, stabilizes lysosomes and thereby protects from cell death.Results Treatment with cholesterol modifying drugs results in alterations of the lysosomal compartmentWe hypothesized that modulation of lysosomal composition affects cellular sensitivity to apoptosis and tested our theory using human fibroblasts derived from a patient with NPC. The cells harbor mutations in the NPC1 gene and have a negligible expression of the NPC1 protein (results not shown). Cellular cholesterol levels were 2-fold higher in NPC1-mutant fibroblasts compared with wild type (wt) fibroblasts (Figure 1A). Labeling of unesterified cholesterol using the antibiotic filipin showed a perinuclear vesicular pattern in NPC1-mutant cells, confirming that cholesterol accumulated in late endosomes/lysosomes (Figure 1B). Cholesterol levels were manipulated in these two cell models, by agents that were reported to change the lipid composition of lysosomes. Treatment of wt fibroblasts with U18666A or quinacrine resulted in cholesterol accumulation, as assessed by analysis of unesterified cholesterol content and lysosomal location was confirmed by filipin staining (Figure 1A and B). In addition, both agents induced expansion of the lysosomal compartment, as demonstrated by increased Lysotracker fluorescence (Figure 1C and E). NPC1-mutant fibroblasts were treated with methyl-b-cyclodextrin (MbCD) and 25-HC, substances suggested to revert cholesterol storage. The results verified that both age.Nt protects cells from LMP-dependent apoptosis [20]. Although this finding, which has also been confirmed by others [21], may seem counterintuitive, it is possibleLysosomal Stability Is Regulated by CholesterolFigure 2. Manipulation of lysosomal cholesterol content modulates the cellular sensitivity to apoptosis. Cholesterol content of human fibroblasts was modulated using U18666A, quinacrine, methyl-b-cyclodextrin (MbCD) or 25-hydroxy cholesterol (25-HC) before apoptosis was induced using O-methyl-serine dodecylamide hydrochloride (MSDH; 24 h). Phase contrast images of A) wt and B) NPC1-mutant fibroblasts (NPC1mut). Scale bar 20 mm. C) Viability of cultures in A and B, respectively, assessed by the MTT assay (n = 4). Viability is expressed as percentage of untreated cultures. D) Caspase-3 like activity (n = 4?). E) Representative curve of increase in green fluorescence during photo-oxidation of acridine orange. F) Quantification of lag time (as presented in E; n = 5?). Data are presented as the mean 6 SD, * p#0.05. doi:10.1371/journal.pone.0050262.gthat cholesterol preserves the integrity of the lysosomal membrane and thus promotes neuronal survival upon acute cellular stress. Importantly, in both NPC1-mutant cells and U18666A treated cells, cholesterol accumulation is associated with storage of severalother lipids [9,22], which might influence lysosomal stability. In addition, the expression of LAMP-2 was increased by U18666A treatment [20]. Because LAMPs have been shown to be important for regulation of LMP [23], further studies to distinguish betweenLysosomal Stability Is Regulated by Cholesterolthe LMP-modulating roles of cholesterol, sphingolipids and altered LAMP-1 and 22 expression were undertaken. We hypothesize that modulation of lysosomal composition affects cellular sensitivity to apoptosis and cell fate can be manipulated by the use of agents inducing or reducing cholesterol content. We herein provide evidence that cholesterol, and not accompanying sphingolipids or LAMP proteins, stabilizes lysosomes and thereby protects from cell death.Results Treatment with cholesterol modifying drugs results in alterations of the lysosomal compartmentWe hypothesized that modulation of lysosomal composition affects cellular sensitivity to apoptosis and tested our theory using human fibroblasts derived from a patient with NPC. The cells harbor mutations in the NPC1 gene and have a negligible expression of the NPC1 protein (results not shown). Cellular cholesterol levels were 2-fold higher in NPC1-mutant fibroblasts compared with wild type (wt) fibroblasts (Figure 1A). Labeling of unesterified cholesterol using the antibiotic filipin showed a perinuclear vesicular pattern in NPC1-mutant cells, confirming that cholesterol accumulated in late endosomes/lysosomes (Figure 1B). Cholesterol levels were manipulated in these two cell models, by agents that were reported to change the lipid composition of lysosomes. Treatment of wt fibroblasts with U18666A or quinacrine resulted in cholesterol accumulation, as assessed by analysis of unesterified cholesterol content and lysosomal location was confirmed by filipin staining (Figure 1A and B). In addition, both agents induced expansion of the lysosomal compartment, as demonstrated by increased Lysotracker fluorescence (Figure 1C and E). NPC1-mutant fibroblasts were treated with methyl-b-cyclodextrin (MbCD) and 25-HC, substances suggested to revert cholesterol storage. The results verified that both age.
