Gamma-secretase activity is present in rafts but is not cholesterol-dependent.
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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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 · 4.85 Impact Factor
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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.Biochimica et Biophysica Acta 07/2010; 1802(7-8):682-91. DOI:10.1016/j.bbadis.2010.05.006 · 4.66 Impact Factor
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ABSTRACT: It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and gamma-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Abeta(40) and Abeta(42) in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of gamma-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.Journal of Neurochemistry 05/2009; 110(1):220-30. DOI:10.1111/j.1471-4159.2009.06126.x · 4.24 Impact Factor