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.
- Future Lipidology - FUTURE LIPIDOL. 01/2006; 1(4):441-453.
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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; · 5.94 Impact Factor
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ABSTRACT: Brains of patients affected by Alzheimer's disease (AD) contain large deposits of aggregated amyloid β-protein (Aβ). Only a small fraction of the amyloid precursor protein (APP) gives rise to Aβ. Here, we report that ∼10% of APP undergoes a post-translational lipid modification called palmitoylation. We identified the palmitoylation sites in APP at Cys(186) and Cys(187). Surprisingly, point mutations introduced into these cysteines caused nearly complete ER retention of APP. Thus, either APP palmitoylation or disulfide bridges involving these Cys residues appear to be required for ER exit of APP. In later compartments, palmitoylated APP (palAPP) was specifically enriched in lipid rafts. In vitro BACE1 cleavage assays using cell or mouse brain lipid rafts showed that APP palmitoylation enhanced BACE1-mediated processing of APP. Interestingly, we detected an age-dependent increase in endogenous mouse brain palAPP levels. Overexpression of selected DHHC palmitoyl acyltransferases increased palmitoylation of APP and doubled Aβ production, while two palmitoylation inhibitors reduced palAPP levels and APP processing. We have found previously that acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition led to impaired APP processing. Here we demonstrate that pharmacological inhibition or genetic inactivation of ACAT decrease lipid raft palAPP levels by up to 76%, likely resulting in impaired APP processing. Together, our results indicate that APP palmitoylation enhances amyloidogenic processing by targeting APP to lipid rafts and enhancing its BACE1-mediated cleavage. Thus, inhibition of palAPP formation by ACAT or specific palmitoylation inhibitors would appear to be a valid strategy for prevention and/or treatment of AD.Journal of Neuroscience 07/2013; 33(27):11169-83. · 6.91 Impact Factor