Cholesterol retention in Alzheimer's brain is responsible for high β- and γ-secretase activities and Aβ production

Abstract

Alzheimer's disease (AD) is characterized by overproduction of Aβ derived from APP cleavage via β- and γ-secretase pathway. Recent evidence has linked altered cholesterol metabolism to AD pathogenesis. In this study, we show that AD brain had significant cholesterol retention and high β- and γ-secretase activities as compared to age-matched non-demented controls (ND). Over one-half of AD patients had an apoE4 allele but none of the ND. β- and γ-secretase activities were significantly stimulated in vitro by 40 and 80 μM cholesterol in AD and ND brains, respectively. Both secretase activities in AD brain were more sensitive to cholesterol (40 μM) than those of ND (80 μM). Filipin-stained cholesterol overlapped with BACE and Aβ in AD brain sections. Cholesterol (10–80 μM) added to N2a cultures significantly increased cellular cholesterol, β- and γ-secretase activities and Aβ secretion. Similarly, addition of cholesterol (20–80 μM) to cell lysates stimulated both in vitro secretase activities. Ergosterol slightly decreased β-secretase activity at 20–80 μM, but strongly inhibited γ-secretase activity at 40 μM. Cholesterol depletion reduced cellular cholesterol, β-secretase activity and Aβ secretion. Transcription factor profiling shows that several key nuclear receptors involving cholesterol metabolism were significantly altered in AD brain, including decreased LXR-β, PPAR and TR, and increased RXR. Treatment of N2a cells with LXR, RXR or PPAR agonists strongly stimulated cellular cholesterol efflux to HDL and reduced cellular cholesterol and β-/γ-secretase activities. This study provides direct evidence that cholesterol homeostasis is impaired in AD brain and suggests that altered levels or activities of nuclear receptors may contribute to cholesterol retention which likely enhances β- and γ-secretase activities and Aβ production in human brain.

Introduction

Alzheimer’s disease (AD) is characterized by overproduction and deposition of β-amyloid peptides (Aβ) in the brain. Aβ peptide is derived from proteolytic cleavage of β-amyloid precursor protein (APP). Three secretases, α, β, and γ, are involved in APP processing (Walter et al., 2001, Selkoe, 2004). Sequential cleavage of APP by β- and γ-secretases yields either Aβ_1–40 or Aβ_1–42 peptide (Walter et al., 2001, Selkoe, 2004); whereas sequential α- and γ-secretase cleavage of APP does not generate Aβ. It is unknown which factor(s) determines the switch between the two APP processing pathways. Recent studies have linked altered cholesterol metabolism and increased Aβ production to AD pathogenesis (Miller and Chacko, 2004, Wolozin, 2001, Wolozin, 2004, Puglielli et al., 2005). Epidemiology studies have found that hypercholesterolemia is an early risk factor of AD, and decreased prevalence of AD is associated with use of cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase inhibitors or statins) (Wolozin et al., 2000, Wolozin, 2004, Tan et al., 2003, Puglielli et al., 2005). Treatment with satins reduced serum Aβ peptides in vivo and the release of Aβ from cultured cells (Refolo et al., 2001, Sjogren et al., 2003, Friedhoff et al., 2001, Kojro et al., 2001). Experimental studies have shown that hypercholesterolemia accelerated AD pathology (Refolo et al., 2000), and cholesterol-lowering drugs reduced Aβ pathology in transgenic animal models (Refolo et al., 2001). All of these studies suggest a close correlation between cholesterol and AD pathogenesis. Cholesterol may thus be one of the factors that determine APP processing pathways and Aβ production in AD.

Since α-secretase is located in phospholipid-rich domains and both β- and γ-secretases reside in cholesterol-rich lipid rafts of plasma membrane (Wahrle et al., 2002, Cordy et al., 2003), it is believed that altered levels of cholesterol or/and ratio of cholesterol to phospholipids in cellular membrane could affect secretase activities and determine preferential APP processing pathways (Miller and Chacko, 2004, Wolozin, 2004, Kaether and Haass, 2004). Wolozin hypothesized that high cholesterol inhibits α-secretase and promotes β- and γ-secretase activities (Wolozin, 2001, Wolozin, 2004); while Kaether and Haass (2004) proposed that a moderate low-level of cholesterol increases Aβ production; whereas extremely poor-cholesterol environment inhibits Aβ production. Experimental studies yielded contradictory findings about cholesterol and Aβ generation. Simons et al. (1998) reported that cholesterol depletion inhibited Aβ production in hippocampal neurons. Cordy et al. (2003) showed that Aβ secretion was reduced when lipid rafts were disrupted by depleting cellular cholesterol. In another study, cloned β-secretase was purified and reconstituted in vitro with liposomes (Kalvodova et al., 2005). The study found that neutral glycosphingolipids, anionic glycerophospholipids, and cholesterol stimulated β-secretase activity. Wahrle et al. (2002) found that γ-secretase activity was located in cholesterol-rich membrane microdomains and was cholesterol-dependent in that cholesterol depletion inhibited γ-secretase activity and cholesterol replacement restored its activity. In contrary, Abad-Rodriguez et al. (2004) reported that cholesterol loss in hippocampal neurons enhanced Aβ generation. These studies provide the evidence that cholesterol affects β- and γ-secretase activities in cultured cells or reconstituted systems. However, little is known whether cholesterol metabolism and its regulation are altered in AD brain and whether cholesterol can regulate β- and γ-secretase activities in human brain tissue. Several studies suggested that nuclear receptors LXR or RXR affect the expression of a cholesterol transporter ABCA1 and therefore Aβ production in cultured cells or animals but these studies yielded controversial results (Fukumoto et al., 2002, Koldamova et al., 2003, Koldamova et al., 2005a, Koldamova et al., 2005b, Sun et al., 2003). Nevertheless, there is no evidence whether levels or activities of these nuclear receptors are altered in AD brain or not. We hypothesize that cholesterol homeostasis and its regulatory mechanism are impaired in AD brain where the environment enhances β- and γ-secretase activities and APP is processed primarily via β- and γ-secretase pathway. In this report, we provide direct evidence that cholesterol metabolism is altered in AD brain and this alteration is typified by significant cholesterol retention and high levels of β- and γ-secretase activities. The study demonstrates that cholesterol can regulate β- and γ-secretase activities in human brain and cultured cells. Altered levels or activities of nuclear receptors are likely responsible for impaired cholesterol homeostasis and consequent increases in β- and γ-secretase activities and Aβ production in AD brain.