Ic to neurons, and cholesterol accumulation actually protected neurons from (��)-Imazamox apoptosis induced by MSDH and oxidative stress. These results might seem contradictory, as NPC is a chronic neurodegenerative disease (i.e., associated with neuronal death). The reason for the neuronal vulnerability has not been elucidated, but neurons seem to be particularly susceptible to disturbances of lysosomal function [35,36], and cholesterol storage in lysosomes induces additional changes in the lysosomal system. In the brains of NPC12/2 mice, increased levels of cathepsins have been demonstrated [37]. Furthermore, in NPC-mutant cells, fusion and fission of late endosomes and lysosomes are reduced [38], and vesicle trafficking is impaired [39,40]. Although cholesterol accumulation confers protection toward acute stress, it remains likely that the associated additional disturbances in lysosomal function may have deleterious effects in the cell in the long run. Noteworthy, disruption of the lysosomal system is implicated in the development of many neurodegenerative disorders that also have a connection to altered cholesterol homeostasis, such as Alzheimer’s, Parkinson’s and Huntington’s diseases [35]. These disorders are characterized by selective vulnerability of specific brain areas to neurodegeneration and oxidative stress [41]. Interestingly, in cells adapted to chronic oxidative stress, resistance was associated with intracellular cholesterol accumulation. Analysis of brain tissue reveals that stress-resistant cells in vitro showed similar features to the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area [42]. These results highlight the possibility that alterations in membraneLysosomal Stability Is Regulated by CholesterolFigure 5. Cholesterol Accumulation Protects MEFs from Oxidative Stress-induced Apoptosis, Independent 1531364 of the Expression of LAMP Proteins. A) Localization (scale bar 10 mm) and B) expression of lysosome-associated membrane protein-2 (LAMP-2) in wt and NPC1-mutant (NPC1mut) human fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to MNS site verify equal protein loading. One representative blot out of three is shown. C) Phase contrast images (scale bar 5 mm) and D) viability analysis (n = 4) of wt mouse embryonic fibroblasts (MEFs) and MEFs deficient for LAMP-1 (LAMP-12/2) or LAMP-2 (LAMP-22/2) 24 h after H2O2 exposure, with or without U18666A pretreatment. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean 6 SD, * p#0.05) Filipin staining in MEFs, with or without U18666A treatment. Scale bar 10 mm. doi:10.1371/journal.pone.0050262.gcholesterol composition may be at least partly involved in the responses allowing neurons to cope with prolonged stress. Several factors have been suggested to be involved in the regulation of lysosomal stability, such as the lipid composition of the lysosomal membrane as well as lysosomal membrane proteins. Our results demonstrate that the manipulation of lysosomal cholesterol content can be used to modify apoptosis sensitivity. Our data indicate that short-term lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.Cells and culture conditionsWt (GM05659) and NPC1-mutant (GM18436) human fibroblasts (passages 12?4; Coriell Institute, Camden, NJ, USA) were cultured in E.Ic to neurons, and cholesterol accumulation actually protected neurons from apoptosis induced by MSDH and oxidative stress. These results might seem contradictory, as NPC is a chronic neurodegenerative disease (i.e., associated with neuronal death). The reason for the neuronal vulnerability has not been elucidated, but neurons seem to be particularly susceptible to disturbances of lysosomal function [35,36], and cholesterol storage in lysosomes induces additional changes in the lysosomal system. In the brains of NPC12/2 mice, increased levels of cathepsins have been demonstrated [37]. Furthermore, in NPC-mutant cells, fusion and fission of late endosomes and lysosomes are reduced [38], and vesicle trafficking is impaired [39,40]. Although cholesterol accumulation confers protection toward acute stress, it remains likely that the associated additional disturbances in lysosomal function may have deleterious effects in the cell in the long run. Noteworthy, disruption of the lysosomal system is implicated in the development of many neurodegenerative disorders that also have a connection to altered cholesterol homeostasis, such as Alzheimer’s, Parkinson’s and Huntington’s diseases [35]. These disorders are characterized by selective vulnerability of specific brain areas to neurodegeneration and oxidative stress [41]. Interestingly, in cells adapted to chronic oxidative stress, resistance was associated with intracellular cholesterol accumulation. Analysis of brain tissue reveals that stress-resistant cells in vitro showed similar features to the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area [42]. These results highlight the possibility that alterations in membraneLysosomal Stability Is Regulated by CholesterolFigure 5. Cholesterol Accumulation Protects MEFs from Oxidative Stress-induced Apoptosis, Independent 1531364 of the Expression of LAMP Proteins. A) Localization (scale bar 10 mm) and B) expression of lysosome-associated membrane protein-2 (LAMP-2) in wt and NPC1-mutant (NPC1mut) human fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to verify equal protein loading. One representative blot out of three is shown. C) Phase contrast images (scale bar 5 mm) and D) viability analysis (n = 4) of wt mouse embryonic fibroblasts (MEFs) and MEFs deficient for LAMP-1 (LAMP-12/2) or LAMP-2 (LAMP-22/2) 24 h after H2O2 exposure, with or without U18666A pretreatment. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean 6 SD, * p#0.05) Filipin staining in MEFs, with or without U18666A treatment. Scale bar 10 mm. doi:10.1371/journal.pone.0050262.gcholesterol composition may be at least partly involved in the responses allowing neurons to cope with prolonged stress. Several factors have been suggested to be involved in the regulation of lysosomal stability, such as the lipid composition of the lysosomal membrane as well as lysosomal membrane proteins. Our results demonstrate that the manipulation of lysosomal cholesterol content can be used to modify apoptosis sensitivity. Our data indicate that short-term lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.Cells and culture conditionsWt (GM05659) and NPC1-mutant (GM18436) human fibroblasts (passages 12?4; Coriell Institute, Camden, NJ, USA) were cultured in E.
