Cholesterol retention in Alzheimer's brain is responsible for high β- and γ-secretase activities and Aβ production
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 Aβ1–40 or Aβ1–42 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.
Section snippets
Cell cultures
Mouse neuroblastoma 2a (N2a) and K269 cells stably transfected with a human APP gene were generously provided by Dr. Huaxi Xu at the Burnham Institute and Dr. Dennis Selkoe at the Harvard Medical School, respectively. N2a cells were grown in media containing 50% Dulbecco’s Modified Eagle’s Medium (DMEM), 50% Opti-MEM, 1× penicillin–streptomycin, 200 μg/ml G418 (GIBCO/Invitrogen, Burlington, ON), and 10% fetal bovine serum (FBS) (HyClone, Logan, UT). K269 cells were grown in DMEM containing 10%
Patients’ information, immunostaining, and APOEε4 allele
The average age of ND and AD patients was 88.08 ± 5.24 and 83.85 ± 7.02 years, respectively. For 12 ND cases, there were 6 males and 6 females with an average age of 88.83 ± 13.2 and 87.33 ± 5.35 years, respectively. For 13 AD patients, there were 10 males and 3 female with an average age of 82.4 ± 6.8 and 88.67 ± 6.51 years, respectively.
Anti-Aβ staining was performed on brain tissue samples to confirm the absence or presence of Aβ peptide deposition in ND and AD brain samples, respectively. Large amounts
AD brain exhibited significant cholesterol retention and high levels of β- and γ-secretase activities
Cholesterol has been suggested to be a risk factor of AD and to affect β- and γ-secretase activities in cells. Several experimental studies support the hypothesis (Wahrle et al., 2002, Cordy et al., 2003, Kalvodova et al., 2005, Abad-Rodriguez et al., 2004). However, little is known whether the levels of cholesterol or β- and γ-secretase activities are altered in AD brain as compared to ND brain tissues. Cholesterol measurement by the enzymatic assay showed that the level of cholesterol content
Cholesterol increased β- and γ-secretase activities and Aβ1–40 production in N2a cells
In order to confirm the observations on cholesterol and secretase activities obtained from human brain tissues, we studied whether cholesterol added to culture media could affect β- and γ-secretase activities in N2a cells overexpressing hAPP. Cholesterol added to culture media can be incorporated into cell as shown by cholesterol labeling of K269 cells and efflux assays by us (see below) and others (Fukumoto et al., 2002, Koldamova et al., 2003, Hirsch-Reinshagen et al., 2004, Wang et al., 2004
Cholesterol depletion reduced cellular cholesterol, β-secretase activity and Aβ1–40 production in N2a cells
To determine whether cholesterol depletion would affect secretase activity and Aβ production, N2a cells were treated with 2.5 mM β-methyl cyclodextran (CDT) for 2 h. The media containing CDT were removed, cells washed twice and fresh complete media were added. The cells and media were then harvested at 4, 8, 12, and 24 h thereafter for cholesterol assays, in vitro β-secretase activity assay, and Aβ1–40 ELISA. Cellular cholesterol was significantly reduced at 4 h post-treatment (One-way ANOVA, p =
Expression levels of cholesterol efflux transporters ABCA1 and ABCG1 in ND and AD
ABCA1 is a large transmembrane protein and mediates cellular cholesterol efflux to apolipoproteins. Several in vitro studies suggested that ABCA1-mediated cholesterol efflux affect APP processing and Aβ production in cells (Fukumoto et al., 2002, Koldamova et al., 2003, Koldamova et al., 2005a, Koldamova et al., 2005b). In order to explore the link between ABCA1 and AD, we examined the expression of ABCA1 at both the mRNA and protein levels in ND and AD brain tissues. Real-time qRT-PCR showed
Transcription factors involved in lipid metabolism were altered in AD brains
Our above experiments showed that cholesterol metabolism and homeostasis was altered in AD brain, resulting in significant cholesterol retention. A number of studies have shown that nuclear receptors play important roles in the regulation of cholesterol and lipid metabolism and in maintaining cholesterol homeostasis in cells and tissues (Schmitz and Langmann, 2005, Edwards et al., 2002, Francis et al., 2003). Nuclear receptors (NR) are ligand-inducible transcription factors composed of a
LXR, RXR and PPAR agonists reduced cellular cholesterol and β- and γ-secretase activities in cells
LXR, RXR and PPAR are principal nuclear receptors that regulate the metabolism of lipids and cholesterol. A number of studies have shown that LXR or/and RXR agonists can up-regulate the expression of lipid/cholesterol metabolism-related genes and ABCA1 and therefore reduce cellular cholesterol (Fukumoto et al., 2002, Koldamova et al., 2003, Sun et al., 2003, Wang et al., 2004, Schmitz and Langmann, 2005). One study showed that LXR agonists increased ABCA1 expression and Aβ production in neural
Discussion
Altered cholesterol metabolism has been linked to AD pathogenesis. However, little is known whether cholesterol metabolism is altered in AD brain or not. This study provides direct evidence that altered cholesterol metabolism does exist in AD brain. There are two APP processing pathways, α- and γ-secretase and β- and γ-secretase pathways. Cholesterol may serve as a determinant for switching the two pathways. This is supported by the findings that AD brains had significant cholesterol retention
Acknowledgments
The authors thank Dr. Dennis Selkoe at the Harvard Medical School for providing K269 cells for the study and Ms. Ewa Baumann for doing some of the brain tissue sections and staining. The study was supported by funding from a Canadian Research Program “Vascular Health & Dementia” sponsored by Heart & Stroke Foundation of Canada, Canadian Institutes of Health Research, Alzheimer Society of Canada, and Pfizer.
References (63)
- et al.
The gene encoding nicastrin, a major gamma-secretase component, modifies risk for familial early-onset Alzheimer disease in a Dutch population-based sample
Am. J. Hum. Genet.
(2002) - et al.
BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis
J. Lipid Res.
(2002) - et al.
Induction of the cholesterol transporter ABCA1 in central nervous system cells by liver X receptor agonists increases secreted Abeta levels
J. Biol. Chem.
(2002) - et al.
Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain
J. Biol. Chem.
(2004) - et al.
The absence of ABCA1 decreases soluble apoE levels but does not diminish amyloid deposition in two murine models of Alzheimer's disease
J. Biol. Chem.
(2005) - et al.
Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI
J. Lipid Res.
(1990) - et al.
Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro
J. Biol. Chem.
(2005) - et al.
22R-hydroxycholesterol and 9-cis-retinoic acid induce ATP-binding cassette transporter A1 expression and cholesterol efflux in brain cells and decrease amyloid beta secretion
J. Biol. Chem.
(2003) - et al.
Lack of ABCA1 considerably decreases brain ApoE level and increases amyloid deposition in APP23 mice
J. Biol. Chem.
(2005) - et al.
The liver X receptor ligand T0901317 decreases amyloid beta production in vitro and in a mouse model of Alzheimer's disease
J. Biol. Chem.
(2005)
PPARg agonists as new therapeutic agents for the treatment of Alzheimer's disease
Exp. Neurol.
A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity
J. Biol. Chem.
Development of rapid staining protocols for laser capture microdissection of brain vessels from human and rat coupled to gene expression analyses
J. Neurosci. Methods
Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model
Neurobiol. Dis.
A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease
Neurobiol. Dis.
Transcriptional regulatory networks in lipid metabolism control ABCA1 expression
Biochim. Biophys. Acta
Expression of liver X receptor target genes decreases cellular amyloid beta peptide secretion
J. Biol. Chem.
Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains
Neurobiol. Dis.
The cell biology of Alzheimer's disease: uncovering the secrets of secretases
Curr. Opin. Neurobiol.
ABCA1 modulates CSF cholesterol levels and influences the age at onset of Alzheimer's disease
Neurobiol. Aging
Cholesterol and the biology of Alzheimer's disease
Neuron
Increased expression of bioactive chemokines in human cerebromicrovascular endothelial cells and astrocytes subjected to simulated ischemia in vitro
J. Neuroimmunol.
Evidence that hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of interleukin-1β (IL-1β) in astrocyte Cultures
J. Neuroimmunol.
Neuronal membrane cholesterol loss enhances amyloid peptide generation
J. Cell Biol.
Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-d-aspartate receptor redistribution
J. Neurochem.
Conditional disruption of the peroxisome proliferator-activated receptor gamma gene in mice results in lowered expression of ABCA1, ABCG1, and apoE in macrophages and reduced cholesterol efflux
Mol. Cell. Biol.
Brain cholesterol: long secret life behind a barrier
Arterioscler. Thromb. Vasc. Biol.
Exclusively targeting beta-secretase to lipid rafts by GPI-anchor addition up-regulates beta-site processing of the amyloid precursor protein
Proc. Natl. Acad. Sci. U. S. A.
The involvement of lipid rafts in Alzheimer's disease
Mol. Membr. Biol.
Cholesterol metabolism in the brain
Curr. Opin. Lipidol.
Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts
J. Cell Biol.
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