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Function of ß-amyloid in cholesterol transport: a lead to neurotoxicity¹

Division of Hormone Research, Departments of Cell Biology, Pharmacology and Neuroscience, Georgetown University School of Medicine, Washington, D.C., USA

²Correspondence: Division of Hormone Research, Departments of Cell Biology, Pharmacology and Neuroscience, Georgetown University School of Medicine, 3900 Reservoir Road, NW, Washington, DC 20057, USA. E-mail: papadopv{at}georgetown.edu

SPECIFIC AIMS

Amyloid ß-peptide (Aß), Aß precursor protein (APP), apolipoprotein E (apoE), and elevated cholesterol levels have been linked to Alzheimer’s disease (AD) pathology. It is our hypothesis that one of the physiological functions of Aß and APP is to control cholesterol transport. Although AD is associated with increased Aß production, high cholesterol levels also lead to overproduction of Aß. The aim of the present study was to investigate the interaction of cholesterol with Aß, high density lipoproteins (HDL), low density lipoproteins (LDL), and apoE and examine the effect of the Aß–cholesterol interaction on cholesterol trafficking and neuronal function.

PRINCIPAL FINDINGS

1. Cholesterol binds to Aß at the -secretase cleavage site
A novel method, cholesterol-protein binding blot assay (CPBBA), was used to study direct interaction between Aß and cholesterol. The generated pattern shows that after 24 h incubation at 37°C, the intensity of radiolabeling of Aß with ³H-labeled cholesterol decreased in the presence of increasing concentrations of unlabeled cholesterol (Fig. 1 a). The radiolabeled bands were identified as Aß using an antibody specific to Aß (Fig. 1b ); note that despite the decreased radiolabeling of Aß, there are no differences in the amount of Aß present in each lane. These data demonstrate that under native conditions, cholesterol binds to Aß. Using CPBBA, the cholesterol binding site in Aß was mapped and found to be in amino acids 10–20 of Aß (Fig. 1c ). The site where -secretase acts to cleave APP is within the cholesterol binding site (Fig. 1d ). Our data indicate that in the presence of low cholesterol levels APP will be cleaved by -secretase generating the Aß_17–40 peptide. However, in the presence of high cholesterol levels, cholesterol binds to the -secretase cleavage site of APP, thus blocking the action of the enzyme, leading to the generation of Aß_1–40 rather than Aß_17–40.

View larger version (32K): [in this window] [in a new window]   Figure 1. Identification of the Aß–cholesterol binding and binding site by CPBBA and immunoblot analyses. a) 3H-cholesterol (0.15 µCi) only in 20 µl PBS (Ctrl-1); Aß1–42 peptide (50 µM) was incubated with 3H-cholesterol (0.15 µCi) in the absence (Ctrl-2) or presence of increasing concentrations of nonradioactive cholesterol in 20 µl PBS for 24 h at 37°C; Aß1–42 peptide (50 µM) was incubated with 3H-cholesterol (0.15 µCi) in 20 µl PBS for 24 h at 4°C (Ctrl-3). b) Identification of Aß1–42 by a polyclonal rabbit anti-ß-amyloid peptide antiserum on same blot. c) For identifying the cholesterol binding site, Aß peptides (50 µM) were incubated with 3H-cholesterol (0.15 µCi) in 20 µl PBS at 37°C for 24 h. Results shown are representative of 4 indepen-dent experiments. The localization of the cholesterol binding site in Aß is shown in panel d.

2. Aß affects cholesterol-apoE/LDL binding
HDL and LDL are the main carriers of cholesterol that mediate its transport between peripheral tissues and liver and cholesterol influx into the cells. By contrast, apoE is a cholesterol transport protein that mediates cholesterol efflux from cells. In a short (2 h) incubation period and in the absence of apoE, only low binding of cholesterol to Aß_1–40 and Aß_17–40 can be seen (Fig. 2 a). The effect of Aß_17–40 on cholesterol binding to apoE (100 µg protein/ml) was dose dependent with an IC₅₀ of 25 µM (Fig. 2b ). The IC₅₀ of the effect of Aß_1–40 on cholesterol binding to LDL (100 µg protein/ml) was 10 µM (Fig. 2c ). IC₅₀ values for Aß_17–40 and Aß_1–40 depend on the amount of apoE and lipoprotein present in the reaction mixture; decreasing the concentration of apoE and lipoprotein results in lower IC₅₀ values. Aß_17–40 also inhibited in a concentration-dependent manner the binding of cholesterol to three common isoforms of apoE: apoE2 (Fig. 2d ), apoE3 (Fig. 2e ), and apoE4 (Fig. 2f ). In all cases, significant inhibition was observed with 25 µM Aß_17–40. Even at high concentrations, Aß_1–40 did not affect the binding of cholesterol to apoE2, apoE3, and apoE4. These data suggest that in a low cholesterol environment where there is more of the Aß_17–40 peptide, the amount of cholesterol exported by apoE from the cell (cholesterol efflux) decreases, leading to increased intracellular cholesterol levels. In agreement with cholesterol influx data, Aß_17–40 even at high concentrations does not affect cholesterol binding to LDL. However, in the presence of high cholesterol levels, the elevated Aß_1–40 formation does not affect the apoE-mediated cholesterol efflux; rather, the Aß_1–40 generated decreases the amount of cholesterol transported by LDL into the cells (cholesterol influx), leading to decreased cellular cholesterol levels.

View larger version (52K): [in this window] [in a new window]   Figure 2. Effect of increasing concentrations of Aß17–40 and Aß1–40 on apoE and LDL–cholesterol binding examined by CPBBA. No protein (a) and 2 µg of either human apoE (b), LDL (c), apoE2 (d), apoE3 (e), or apoE4 (f) were incubated with 3H-cholesterol (0.1 µCi for apoE or no protein, 0.02 µCi for LDL) in the presence or absence of increasing concentrations of either Aß17–40 or Aß1–40 in 20 µl volume at 37°C for 1 h (for apoE) or 2 h (for LDL). Results shown are representative of 4 independent experiments.

CONCLUSIONS

Despite the large number of studies of the role of APP, apoE, and high cholesterol levels in AD, the detailed molecular mechanisms underlying the actions of these entities leading to the onset and/or progression of the disease have not yet been elucidated. Mutations of APP and PS genes lead to accumulation of Aß and have been described as the leading cause of familial AD. High cholesterol levels also lead to overproduction of Aß and may be responsible for the development of sporadic AD. Elevated cholesterol levels might accentuate the onset and progression of AD due to either APP or PS mutations or other yet unknown factors, leading to either sporadic or familial AD. The possibility that Aß-induced alterations in cellular cholesterol metabolism could contribute to neurodegeneration has been raised and the abnormal lipid composition of cellular membranes in AD brain has been reported. Since growth and maintenance of an elaborate neurite network require directional lipid transport, alterations in lipid transport might be detrimental to neuronal integrity. It has now been established that apoE, a major risk factor in AD, is a component of lipoproteins responsible for the efflux of cholesterol from neurons, apoE2 being more efficacious than apoE3 and apoE4 to mediate cholesterol efflux. Moreover, it has been shown that Aß binds to apoE. Taken together, these results indicate changes in cholesterol transport, and thus homeostasis, as a lead to Aß-mediated neurotoxicity. To examine the role of cholesterol homeostasis in neurodegeneration, we investigated changes induced by low and high cholesterol concentrations on APP, Aß, and apoE levels and have developed a novel method to examine the role of Aß and Aß peptide fragments on apoE and LDL-mediated cholesterol transport, LDL being responsible for cholesterol transfer into the cell.

In the present study, evidence is provided that in the presence of low cholesterol levels and in an environment where Aß exists either normally or is induced by APP or PS mutations, APP will be cleaved by -secretase generating the Aß_17–40 peptide in addition to Aß_1–40. Because there is more of the Aß_17–40 peptide, the amount of cholesterol exported by apoE from the cells decrease, leading to increasing intracellular cholesterol levels.

In the presence of high cholesterol levels, there is increased APP synthesis and processing to Aß and Aß peptide fragments, in agreement with previous reports. Moreover, there is increased apoE synthesis leading to increased cholesterol efflux. Cholesterol binds to the -secretase cleavage site of APP and blocks the action of the enzyme, resulting in generation of Aß_1–40 rather than Aß_17–40. Aß_1–40 does affect either apoE-mediated cholesterol efflux, which is elevated because of increased apoE levels, or the binding of cholesterol to apoE. However, it decreases the amount of cholesterol bound and transported by LDL into the cells, leading to decreased intracellular cholesterol levels. Accumulation of extracellular or membrane free cholesterol will result in neuronal dysfunction. Thus, the presence of excessive levels of cholesterol leads to increased production of Aß, which binds cholesterol and in turn competes against and blocks the LDL-mediated cholesterol influx. Accumulation of extracellular free cholesterol and the subsequent neurotoxicity can be prevented by addition of specific lipoproteins that bind cholesterol in a manner resistant to Aß and Aß peptide fragments. HDL is a lipoprotein that binds cholesterol with higher affinity than apoE (not shown), thus taking cholesterol back to the liver. A schematic representation of cholesterol-induced changes in APP processing and the Aß-induced changes in cholesterol transport is shown in Fig. 3 .

View larger version (38K): [in this window] [in a new window]   Figure 3. Cholesterol-induced mechanism leading to AD pathology. The proposed scheme suggests that cholesterol-induced changes in Aß levels and processing alters lipoprotein- and apoE-mediated cholesterol transport leading to neurotoxicity and AD pathogenesis.

In summary, we propose that one of the physiological functions of Aß and APP is to control cholesterol transport and homeostasis. Overproduction of Aß, due to either mutation of APP and PS genes or high cholesterol levels, blocks cholesterol trafficking, leading to neurodegeneration and the development of familial or sporadic AD pathology.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0285fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0285fje

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