Local cholesterol increase triggers amyloid precursor protein-BACE1 clustering in lipid rafts and rapid endocytosis

Centre de Recherche de l'Institut du Cerveau et de la Moelle, UPMC, INSERM UMR-S975, Centre National de la Recherche Scientifique (CNRS) UMR7225, Hôpital de la Pitié-Salpêtrière, Paris, France.
The FASEB Journal (Impact Factor: 5.04). 03/2011; 25(4):1295-305. DOI: 10.1096/fj.10-168633
Source: PubMed


Amyloid peptide (Aβ) is generated by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Aβ production increases after plasma membrane cholesterol loading through unknown mechanisms. To determine how APP-Bace1 proximity affects this phenomenon, we developed a fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) technique for visualization of these molecules either by epifluorescence or at the plasma membrane only using total internal reflection fluorescence. Further, we used fluorescence correlation spectroscopy to determine the lipid rafts partition of APP-yellow fluorescent protein (YFP) and Bace1-green fluorescent protein (GFP) molecules at the plasma membrane of neurons. We show that less than 10 min after cholesterol exposure, Bace1-GFP/APP-mCherry proximity increases selectively at the membrane and APP relocalizes to raft domains, preceded by rapid endocytosis. After longer cholesterol exposures, APP and Bace1 are found in proximity intracellularly. We demonstrate that cholesterol loading does not increase Aβ production by having a direct impact on Bace1 catalytic activity but rather by altering the accessibility of Bace1 to its substrate, APP. This change in accessibility is mediated by clustering in lipid rafts, followed by rapid endocytosis.

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    • "APP is preferentially processed within cholesterol and sphingolipid-enriched lipid rafts, membrane microdomains where APP encounters b-and g-secretases (Vetrivel and Thinakaran, 2006). APP cleavage by BACE1 and g-secretase produces b-CTF and amyloid-b peptides (amyloid-b 40 and amyloid-b 42 ) (von Arnim et al., 2008; Cossec et al., 2010; Marquer et al., 2011). Figure 3 Neuronal cholesterol accumulation induced by inhibition of Cyp46a1 gene expression triggers endoplasmic reticulum stress. "
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    ABSTRACT: Abnormalities in neuronal cholesterol homeostasis have been suspected or observed in several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, it has not been demonstrated whether an increased abundance of cholesterol in neurons in vivo contributes to neurodegeneration. To address this issue, we used RNA interference methodology to inhibit the expression of cholesterol 24-hydroxylase, encoded by the Cyp46a1 gene, in the hippocampus of normal mice. Cholesterol 24-hydroxylase controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. We used an adeno-associated virus vector encoding short hairpin RNA directed against the mouse Cyp46a1 mRNA to decrease the expression of the Cyp46a1 gene in hippocampal neurons of normal mice. This increased the cholesterol concentration in neurons, followed by cognitive deficits and hippocampal atrophy due to apoptotic neuronal death. Prior to neuronal death, the recruitment of the amyloid protein precursor to lipid rafts was enhanced leading to the production of β-C-terminal fragment and amyloid-β peptides. Abnormal phosphorylation of tau and endoplasmic reticulum stress were also observed. In the APP23 mouse model of Alzheimer's disease, the abundance of amyloid-β peptides increased following inhibition of Cyp46a1 expression, and neuronal death was more widespread than in normal mice. Altogether, these results suggest that increased amounts of neuronal cholesterol within the brain may contribute to inducing and/or aggravating Alzheimer's disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email:
    Brain 07/2015; 138(8). DOI:10.1093/brain/awv166 · 9.20 Impact Factor
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    • "Filipin staining was visualized on a confocal microscope (SP5, Leica Microsystems, Wetzlar, Germany) with excitation at 351 nm by a UV laser. A 9 60 oil-immersion objective of numerical aperture (NA) 1.4 was used to collect light emitted between 400 and 680 nm (Marquer et al., 2011). Specific antibodies were used to detect green fluorescent protein (GFP) in cells expressing the adeno-associated virus (AAV)-vector construction and to recognize neurons and glial cells. "
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    ABSTRACT: Elevations in neuronal cholesterol have been associated with several degenerative diseases. An enhanced excitability and synchronous firing in surviving neurons are among the sequels of neuronal death in these diseases and also in some epileptic syndromes. Here, we attempted to increase neuronal cholesterol levels, using a short hairpin RNA (shRNA) to suppress expression of the enzyme CYP46A1. This protein hydroxylates cholesterol and so facilitates trans-membrane extrusion. A sh-RNA CYP46A1construction coupled to an adeno-associated virus (AAV5) was injected focally and unilaterally into mouse hippocampus. It was selectively expressed first in neurons of the CA3a region. Cytoplasmic and membrane cholesterol increased, neuronal soma volume increased and then decreased before pyramidal cells died. As CA3a pyramidal cells died, inter-ictal EEG events occurred during exploration and non-REM sleep. With time, neuronal death spread to involve pyramidal cells and interneurons of the CA1 region. CA1 neuronal death was correlated with a delayed local expression of phosphorylated tau. Astrocytes were activated throughout the hippocampus and microglial activation was specific to regions of neuronal death. CA1 neuronal death was correlated with distinct aberrant EEG activity. During exploratory behaviour and rapid eye movement sleep, EEG oscillations at 7-10 Hz (theta) could accelerate to 14-21 Hz (beta) waves. They were accompanied by low amplitude, high-frequency oscillations of peak power at ~300Hz and a range of 250-350 Hz. While episodes of EEG acceleration were not correlated with changes in exploratory behaviour, they were followed in some animals by structured seizure-like discharges. These data strengthen links between increased cholesterol, neuronal sclerosis and epileptic behavior. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    European Journal of Neuroscience 04/2015; 41(10). DOI:10.1111/ejn.12911 · 3.18 Impact Factor
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    • "Differences in the treatment regime and animal models used may explain discrepancies between the studies. On the neuronal level, a cholesterol-mediated relocation of APP from the non-lipid raft part of the membranes to the lipid rafts may underlie the stimulatory impact of cholesterol on Aβ generation [135]. This relocation increases the accessibility of β-secretase to its substrate APP and thereby promotes the amyloidogenic cleavage of APP. "
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    ABSTRACT: In mammals, the central nervous system (CNS) is the most cholesterol rich organ by weight. Cholesterol metabolism is tightly regulated in the CNS and all cholesterol available is synthesized in situ. Deficits in cholesterol homeostasis at the level of synthesis, transport, or catabolism result in severe disorders featured by neurological disability. Recent studies indicate that a disturbed cholesterol metabolism is involved in CNS disorders, such as Alzheimer’s disease (AD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). In contrast to circulating cholesterol, dietary plant sterols, can cross the blood-brain barrier and accumulate in the membranes of CNS cells. Plant sterols are well-known for their ability to lower circulating cholesterol levels. The finding that they gain access to the CNS has fueled research focusing on the physiological roles of plant sterols in the healthy and diseased CNS. To date, both beneficial and detrimental effects of plant sterols on CNS disorders are defined. In this review, we discuss recent findings regarding the impact of plant sterols on homeostatic and pathogenic processes in the CNS, and elaborate on the therapeutic potential of plant sterols in CNS disorders. doi 10.1016/j.plipres.2015.01.003
    Progress in Lipid Research 01/2015; 58. DOI:10.1016/j.plipres.2015.01.003 · 10.02 Impact Factor
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