γ-Secretase Activity Is Present in Rafts but Is Not Cholesterol-Dependent

Department of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Biochemistry (Impact Factor: 3.02). 01/2004; 42(47):13977-86. DOI: 10.1021/bi034904j
Source: PubMed

ABSTRACT Cholesterol has been claimed to be involved in the generation and/or accumulation of amyloid beta protein (Abeta). However, the underlying molecular mechanisms have not been fully elucidated yet. Here, we have investigated the effect of membrane cholesterol content on gamma-secretase activity using Chinese hamster ovary cells stably expressing beta-amyloid precursor protein (APP) and either wild-type or N141I mutant-type presenilin 2. Cholesterol was acutely depleted from the isolated membrane by methyl-beta-cyclodextrin, and Abeta production was assessed in a cell-free assay system. Reduced cholesterol did not significantly alter the amounts of Abeta produced by either total cell membranes or cholesterol-rich low-density membrane domains. Even its extremely low levels in the latter domains did not affect Abeta production. This indicates that the membrane cholesterol content does not directly modulate the activity of gamma-secretase. To ascertain that gamma-secretase resides in cholesterol-rich membrane domains, low-density membrane domains were further fractionated with BCtheta (biotinylated theta-toxin nicked with subtilisin Carlsberg protease), which has recently been shown to bind selectively to rafts of intact cells. The membrane domains purified with BCtheta did indeed produce Abeta. These observations indicate that the gamma-cleavage required for generating Abeta occurs in rafts, but its activity is virtually cholesterol-independent.

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    • "Because PS1 has been described as a lipid raft resident protein ( Parkin et al . , 1999 ; Vetrivel et al . , 2004 ; Wada et al . , 2003 ) , and PRiMA can target G 4 AChE to membrane rafts ( Xie et al . , 2010a ) , we investigated whether both PS1 and PRiMA - AChE colocalize to lipid rafts . The distribution of PS1 and AChE was compared by immu - nocytochemistry in H4 cells ( a cell line widely used for lipid raft studies ; Asai et al . , 2007 ; Guardia - Laguarta et al "
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    ABSTRACT: Presenilin-1 (PS1) is the catalytic component of the γ-secretase complex. In this study, we explore if PS1 participates in the processing of the cholinergic acetylcholinesterase (AChE). The major AChE variant expressed in the brain is a tetramer (G4) bound to a proline-rich membrane anchor (PRiMA). Overexpression of the transmembrane PRiMA protein in Chinese hamster ovary cells expressing AChE and treated with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester have enabled us to study whether, through its γ-secretase activity, PS1 participates in the processing of PRiMA-linked AChE. γ-Secretase inhibition led to a notable increase in the level of PRiMA-linked AChE, suggesting that γ-secretase is involved in the cleavage of PRiMA. We demonstrate that cleavage of PRiMA by γ-secretase results in a C-terminal PRiMA fragment. Immunofluorescence labeling allowed us to identify this PRiMA fragment in the nucleus. Moreover, we have determined changes in the proportion of the raft-residing AChE-PRiMA in a PS1 conditional knockout mouse. Our results are of interest as both enzymes have therapeutic relevance for Alzheimer's disease.
    Neurobiology of aging 02/2014; 35(7). DOI:10.1016/j.neurobiolaging.2014.01.147 · 5.01 Impact Factor
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    • "However, these experiments do not reveal a functional context explaining why the APP processing system responds in such a sensitive manner to cholesterol [7]. In addition, multiple experiments demonstrated that the alternation of cholesterol level doesn’t affect the activities of α, β [1], or γ secretase [8], which are critical in APP processing. Thus, more experiments are needed to prove the causal relationship between cholesterol level and APP processing activity. "
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    ABSTRACT: Accumulating experimental evidence support an enhancing effect of free cholesterol on amyloid-beta (Aβ) aggregation. To probe the mechanisms of cholesterol-mediated Aβ aggregation, we applied all-atom molecular dynamic simulations on Aβ42 peptides in presence of free cholesterol. Several control systems were also designed to examine the specificity of cholesterol-residue interactions, including mutation on aromatic residue, substitution of cholesterol with sphingomyelin (SM) and DPPC bilayer, and a mixing SM and cholesterol. Each system was performed 4 independent simulations, with a total time of 560 ns. It was found that cholesterol increased β-sheet formation by 4 folds, but the Phe19→Ser mutation on Aβ42 peptide totally eliminated cholesterol's effect. A stable contact was recognized between the steroid group of cholesterol and the Benzyl group of Phe19. Interestingly, our simulation revealed a regular 1 ns time interval between the establishment of cholesterol-phenylalanine contact and consequent β-sheet formation, suggesting an important role of steroid-benzyl interaction in cholesterol-mediated aggregation. The presence of SM slightly increased β-sheet formation, but the mixture of cholesterol and SM had a strong induction effect. Also, the measurement of Phe19-lipid distance indicates that aromatic side chains of peptides prone to bind to cholesterol on the surface of the mixed micelle. In the DPPC system, polar chains were attracted to the surface of membrane, yielding moderate increase of β-sheet formation. These results shed light on the mechanism of cholesterol-mediated fibrillogenesis, and help to differentiate the effects of cholesterol and other lipids on β-sheet formation process.
    PLoS ONE 09/2012; 7(9):e46245. DOI:10.1371/journal.pone.0046245 · 3.23 Impact Factor
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    • "Elevated dietary cholesterol uptake increased amyloid plaque formation in rabbits and transgenic mice [23] [30], and cholesterol loading and depletion affects Aβ generation in cultured cells [5] [28]. In addition to the indirect effects of cholesterol on APP processing, recent findings suggest that cholesterol may directly modulate the activity of β-secretase (BACE1) [11], while contradictory results have been reported for the effect of cholesterol on γ-secretase cleavage of APP [34] [35]. "
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    ABSTRACT: The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann-Pick type C disease. We found that NPC1(-/-) cells show decreased expression of APP at the cell surface and increased processing of APP through the beta-secretase pathway resulting in increased C99, sAPPbeta and intracellular Abeta40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Abeta/C99 levels in NPC1(-)(/)(-) cells to the levels observed in wt cells. Finding that overexpression of C99, a direct gamma-secretase substrate, does not lead to increased intracellular Abeta levels in NPC1(-)(/)(-) cells vs. CHOwt suggests that the effect on intracellular Abeta upon cholesterol accumulation in NPC1(-)(/)(-) cells is not due to increased APP cleavage by gamma-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and thus its cleavage by beta-secretase.
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