Cholesterol Movement in Niemann-Pick Type C Cells and in Cells Treated with Amphiphiles

Lange, Yvonne; Ye, Jin; Rigney, Mike; Steck, Theodore L.

doi:10.1074/jbc.m000875200

Cited by 170 publications

(207 citation statements)

References 51 publications

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“…Proteins were solubilized with CHAPS, separated by size exclusion chromatography, and the elution profile of Bax was analyzed by immunoblotting. For each condition, even-numbered fractions were loaded on two separate gels; a short exposure time was chosen for the low molecular weight fractions (30-44), and a longer one for the high molecular weight fractions (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) and its sensitivity to trypsin digestion were analyzed by immunoblotting. The presence of tBid was required for the recruitment of Bax to liposomes and led to the appearance of a Tr-Bax fragment (Figure 1d).…”

Section: Resultsmentioning

confidence: 99%

“…U18666A is a drug that induces intracellular cholesterol accumulation, an abnormality that is encountered in Niemann-Pick Type C1 disease. It interferes with the trafficking of cholesterol, causing its increase in late endosomes/lysosomes, 23,24 and inhibits its neosynthesis in the ER. 25 A recent study reported the accumulation of cholesterol in the mitochondria of neurons isolated from an Niemann-Pick Type C1 mouse model, 26 raising the possibility that U18666A might also increase mitochondrial cholesterol content.…”

Section: Resultsmentioning

confidence: 99%

“…23 We therefore ran crude mitochondrial fractions on continuous 8-48% sucrose gradients and analyzed the distribution of mitochondrial proteins by immunoblotting (Figure 3d). Mitochondria isolated from control cells were mainly found in pellets, but those isolated from U18666A-treated cells were more concentrated in fractions corresponding to 48-43% sucrose, indicating that incubation with U18666A led to a significant decrease in the density of mitochondria.…”

Section: Resultsmentioning

confidence: 99%

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Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

2007

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Activation of Bax or Bak is essential for the completion of many apoptotic programmes. Under cytotoxic conditions, these proteins undergo a series of conformational rearrangements that end up with their oligomerization. We found that unlike inactive monomeric Bax, active oligomerized Bax is partially resistant to trypsin digestion, providing a convenient read out to monitor Bax activation. Using this assay, we studied how the lipid composition of membranes affects tBid-induced Bax activation in vitro with pure liposomes. We report that Bax activation is inhibited by cholesterol and by decreases in membrane fluidity. This observation was further tested in vivo using the drug U18666A, which we found increases mitochondrial cholesterol levels. When incubated with tBid, mitochondria isolated from U18666A-treated cells showed a delay in the release of Smac/Diablo and Cytochrome c, as well as in Bax oligomerization. Moreover, pre-incubation with U18666A partially protected cells from stressinduced apoptosis. As many tumours display high mitochondrial cholesterol content, inefficient Bax oligomerization might contribute to their resistance to apoptosis-inducing agents. Among the stimuli that lead to apoptosis, a series of stress conditions trigger the permeabilization of the mitochondrial outer membrane (MOM) and the release of pro-apoptotic factors that will activate caspases in the cytosol. This so-called intrinsic apoptotic pathway is tightly regulated by proteins of the Bcl-2 family, among which the pro-apoptotic members Bax and Bak are essential. They seem to be the central players whose activation results in MOM permeabilization.Under resting conditions, whereas Bak essentially resides in the MOMs, Bax is found in loose association with membranes (mainly the MOMs) or as a soluble protein in the cytosol. Following many cytotoxic signals, Bax and Bak undergo a series of conformational rearrangements. 1 Bax translocates to mitochondria and inserts into the MOMs in a form that cannot be detached by alkali treatment. Bax and Bak then uncover N-terminal epitopes and oligomerize. Oligomerization, shown by size exclusion chromatography, 2 crosslinking experiments 3,4 and native gel electrophoresis, 5 appears as the critical step resulting in MOM permeabilization. However, despite many efforts, it remains unclear how each of these conformational rearrangements is regulated, and how Bax/Bak oligomerization leads to the release of mitochondrial apoptogenic factors. Both processes are undoubtedly regulated by other members of the Bcl-2 family, 6,7 but the participation of many unrelated proteins was also proposed. 8 While a BH3-only protein appears to be sufficient for Bax to oligomerize in synthetic liposomes, 9,10 additional proteins could be required for its activation in isolated mitochondria. 11 These might directly interact with Bax, and/or modify the membrane properties to allow its oligomerization. Interaction with mitochondrial lipids is indeed an important parameter to consider as the final conformational rearrange...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

2007

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“…The main features of the NPC phenotype can be mimicked by cultured cells exposed to a variety of reagents called class 2 amphiphiles, such as U18666A (17). It is now believed that these reagents act directly on the NPC1 protein (18). Based on these facts, we hypothesized that intracellular A␤ metabolism might be changed in these cholesterol-perturbed cells.…”

mentioning

confidence: 99%

Accumulation and Aggregation of Amyloid β-Protein in Late Endosomes of Niemann-Pick Type C Cells

Yamazaki¹,

Chang²,

Haass³

et al. 2001

Journal of Biological Chemistry

156

104

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There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid ␤-protein (A␤) metabolism. However, the precise cellular linkage sites between cholesterol and A␤ have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable A␤ accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This A␤ accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of A␤42 and insolubility in SDS, suggesting the presence of aggregated A␤ in late endosomes. Most importantly, A␤ accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated A␤42 as well as cholesterol, suggesting a direct interaction between aggregated A␤ and cholesterol.Formation of senile plaques composed of amyloid ␤-protein (A␤) 1 is one of the hallmarks of Alzheimer's disease (AD) (1). A␤, a small protein of M r ϳ4,000, is cleaved out sequentially with ␤-and ␥-secretases from a large membrane protein called ␤-amyloid precursor protein (APP), and secreted into the extracellular space. There are two major species of A␤ that are defined by their C-terminal lengths; A␤40 terminates at Val-40, and A␤42 terminates at Ala-42. The latter has a much stronger tendency to aggregate into fibrils and accounts for less than 10% of secreted A␤. However, A␤42 is the most predominant form found in senile plaques and is considered the initial species to be deposited in aged and AD brains (2, 3).Three causative genes, APP, presenilin 1 (PS1), and PS2 have been identified in early onset familial AD (1), and their mutations appear to converge on an increased production of A␤42. However, the pathological mechanisms of sporadic AD, explaining more than 90% of AD patients, remain unknown. Interestingly, there is growing evidence for a significant linkage between A␤ and cholesterol metabolism. First, cholesterol loading or depletion affects A␤ generation both in vitro (4,5) and in vivo (6). Second, the levels of total cholesterol and LDL-cholesterol, but not those of HDL-cholesterol, in the serum correlate with the amount of A␤42 in AD brains (7). Third, aggregated A␤ preferentially binds to cholesterol in vitro (8). Fourth, a substantial fraction of intracellular A␤ is localized in the detergent-insoluble membrane domain that is rich in glycosphingolipid and cholesterol (9, 10). Fifth, the presence of an E4 isoform of apolipoprotein E, an essential molecule for cholesterol metabolism, is a strong risk factor for developing AD (11). Sixth, high dietary cholesterol accelerates AD-related pathologies, including A␤ deposition, in a transgenic mouse model (12). However, no detailed mechanisms for clarifying ...

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“…The increased cholesterol impaired insulin signaling and was restored following cholesterol depletion (24). In other studies cholesterol levels have been reported to be increased, decreased, or unchanged (25)(26)(27)(28).…”

Section: Discussionmentioning

confidence: 88%

Caveolin-associated Accumulation of Globotriaosylceramide in the Vascular Endothelium of α-Galactosidase A Null Mice

Shu

Shayman

2007

Journal of Biological Chemistry

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Cholesterol Movement in Niemann-Pick Type C Cells and in Cells Treated with Amphiphiles

Cited by 170 publications

References 51 publications

Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

Accumulation and Aggregation of Amyloid β-Protein in Late Endosomes of Niemann-Pick Type C Cells

Caveolin-associated Accumulation of Globotriaosylceramide in the Vascular Endothelium of α-Galactosidase A Null Mice

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