Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

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Abstract

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer’s disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1−/− cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP–CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer’s disease and supports the role of lipid rafts in these processes.

Introduction

Formation of amyloid-β peptide (Aβ) is considered to be a central event in the pathogenesis of Alzheimer’s disease (AD) [1], [2]. Aβ is generated through the β-secretase pathway that involves sequential cleavage of β-amyloid precursor protein (APP) by β-secretase (BACE1) followed by γ-secretase (consisting of presenilin 1 (PS1), nicastrin, Aph1 and Pen2). Processing of APP by β-secretase generates a membrane bound C-terminal APP-fragment C99/CTFβ that is further cleaved by γ-secretase to generate Aβ. In parallel, APP can be processed through the non-amyloidogenic α-secretase pathway which involves a sequential cleavage of APP by α- and γ-secretase. Although the details of APP processing are well understood, it is still not known how the partitioning of APP processing through the α- and β-secretase pathway is regulated and what may trigger APP-cleavage. Indeed, it has been postulated that increasing α-secretase processing of APP and/or decreasing β-secretase cleavage in parallel, may be considered for designing novel therapies against Alzheimer’s disease.

Recently, lipid rafts, cholesterol and sphingolipid rich membrane microdomains, have been implicated in the pathogenesis of Alzheimer’s disease [3]. It has been shown that lipid rafts serve as a site of Aβ production [4] and that all three key proteins involved in Aβ formation (APP, BACE1 and γ-secretase complex components) are localized in lipid rafts [5], [6], [7], [8]. The role of lipid rafts in AD has also been supported by evidence that cholesterol may contribute to the pathogenesis of Alzheimer’s disease [9]. It has been hypothesized that cholesterol levels may modulate formation of Aβ by regulating partitioning of APP, BACE1 and/or PS1 to lipid rafts. Indeed, it has been shown that cholesterol depletion disrupts APP [5], BACE1 [6] and PS1 [7] compartmentalization within lipid rafts, leading to decreased Aβ. In contrast, increased lipid raft localization of APP/BACE1 by either antibody co-patching [4] or GPI-anchoring [10] caused increased β-secretase processing of APP and Aβ formation. Overall, these studies suggest that lipid raft compartmentalization of APP, BACE1 and/or PS1 may be an important event in regulating amyloidogenic processing of APP leading to Aβ.

The link between cholesterol and Aβ has been revealed in Niemann Pick type C (NPC) disease as well as AD [11], [12], [13], [14], [15], [16]. NPC1 dysfunction causes accumulation of free cholesterol in late endosomal/lysosomal compartments that leads to increased C99/Aβ formation [13], [14], [16] and a shift in PS1 localization towards early/late endosomes [14], [15]. The aim of this work was to assess whether the cholesterol-effect on C99/Aβ in NPC disease is mediated through lipid rafts. We hypothesized that increased levels of C99/Aβ upon cholesterol accumulation in NPC disease are due to increased compartmentalization of APP in lipid rafts. Indeed, it has been show that excessive storage of cholesterol in lysosomes of NPC1-mutant cells/NPC fibroblasts was accompanied by increased partitioning of cholesterol in lipid rafts [17]. Through these studies we aim to elucidate the role of lipid rafts on APP processing and C99/Aβ production in NPC disease. Our findings may also shed light on the link between cholesterol, lipid rafts and the pathogenesis of Alzheimer’s disease.

Section snippets

Materials and methods

Cell lines, cell tissue culture and transient transfection. Chinese hamster ovary wild type cells (CHOwt) and CHO NPC1-null cells (NPC1−/−, kindly provided by Dr. Daniel Ory) were grown in DMEM:F12 medium (1:1) containing 0.5 mM Na-pyruvate supplemented with 10% FBS, 2 mM l-glutamine and antibiotic/antimycotic solution (all from Invitrogen).

For experiments with γ-secretase inhibitor, cells were treated with 1 μM DAPT in media 24 h before lipid raft isolation.

For transient expression, cells were

Lipid raft isolation – 1% Triton X-100 enables clear separation of lipid raft from non-raft fractions

To investigate whether the cholesterol-effect on APP processing upon NPC1 dysfunction is mediated through lipid rafts we analyzed APP compartmentalization in lipid rafts by sucrose density gradient in CHOwt and CHO NPC1−/− cells. In agreement with previous studies [23], [24], CHO NPC1−/− cells exert NPC-like phenotype (Fig. 1): they show 2-fold increase in free cholesterol levels (Fig. 1A), cholesterol accumulation in punctuate endocytic structures (Fig. 1B) and misstrafficking of

Discussion

Lipid rafts could be a missing link between cholesterol and Alzheimer’s disease (AD). The importance of these cholesterol-rich microdomains on APP processing has been highlighted by the fact that three key proteins in the pathogenesis of AD (APP, BACE1 and PS1) are found in lipid rafts and that cholesterol depletion disrupts this association leading to decreased Aβ[5], [6], [7], [8]. Niemann Pick type C disease (NPC), a lysosomal storage disorder in which cholesterol accumulation in late

Conclusions

We show that cholesterol accumulation upon NPC1 dysfunction leads to increased partitioning of APP/CTFs to lipid rafts. Finding significantly increased levels of CTFs in lipid rafts of NPC1−/− cells vs. CHOwt under γ-secretase inhibitor treatment further indicates that aberrant production of Aβ upon NPC1 dysfunction most likely occurs in lipid rafts. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer’s disease and supports the role of

Acknowledgments

We would like to thank Dr. Daniel Ory for kindly providing CHO NPC1−/− cells and parental CHOwt cells. In addition, we would like to acknowledge Dr. Christian Haass for his generous gift of the C-terminal APP antibody 6687. This work was funded by the Grants: NIH-FIRCA1R03TW007335-01 (to A.G.) and Ministry of Science, Education and Sports of the Republic of Croatia098-0982522-2525 (to S.H.).

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