A novel action of alzheimer's amyloid beta-protein (Abeta): oligomeric Abeta promotes lipid release

Abstract

Interactions between amyloid beta-protein (Abeta) and lipids have been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these interactions has not been fully understood. We examined the effect of Abeta on lipid metabolism in cultured neurons and astrocytes and found that oligomeric Abeta, but not monomeric or fibrillar Abeta, promoted lipid release from both types of cells in a dose- and time-dependent manner. The main components of lipids released after the addition of Abeta were cholesterol, phospholipids, and monosialoganglioside (GM1). Density-gradient and electron microscopic analyses of the conditioned media demonstrated that these Abeta and lipids formed particles and were recovered from the fractions at densities of approximately 1.08-1.18 g/ml, which were similar to those of high-density lipoprotein (HDL) generated by apolipoproteins. The lipid release mediated by Abeta was abolished by concomitant treatment with Congo red and the PKC inhibitor, H7, whereas it was not inhibited with N-acetyl-l-cysteine. These Abeta-lipid particles were not internalized into neurons, whereas HDL-like particles produced by apolipoprotein E were internalized. Our findings indicate that oligomeric Abeta promotes lipid release from neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function.

Fig. 1.

Characterization of Aβ1–40. Aβ1–40 was prepared as described in Materials and Methods. a, The aliquots of iAβ-nonfiltered,iAβ-filtered, fresh Aβ, and PBS were subjected to thioflavin-T assays as described in Materials and Methods. Three independent experiments were performed, and similar results were obtained.b, The equal volume of 2× sample solubilizing buffer was added to each Aβ solution, of which the concentration was normalized with PBS. The samples were then subjected to 4–20% Tris/tricine SDS-PAGE, followed by Western blot analysis.c, Electron micrograph of each sample is shown. The samples were centrifuged at 34,500 rpm for 48 hr using a SW 41-Ti rotor. Electron microscopic analysis of the lower part of each solution containing the resuspended pellet was performed. Results of negative staining show that fibrillar structures are found in the sample ofiAβ-nonfiltered(c); however, no fibril is detected in the samples of iAβ-filtered(d) or fresh Aβ (e). Scale bar, 50 nm.

Fig. 2.

Effect of iAβ on cholesterol and phosphatidylcholine release from neurons in culture. Neuron-rich cultures were labeled with [¹⁴C]acetate for 48 hr as described in Materials and Methods. Cells were then washed three times with DMEM and incubated with iAβ at various concentrations for 4 hr. Synthetic Aβ1–40 dissolved at high concentration and incubated at 37°C for 24 hr, followed by filtration, was used. The released lipids in the media and the cellular lipids were extracted and analyzed as described in Materials and Methods. The iAβ-mediated release of cholesterol and phosphatidylcholine (PC) (a) was significantly increased in a dose-dependent manner. Each data point represents mean ± SE for three samples. For the time course study of iAβ-mediated lipid release from neurons, cultured neurons were labeled with [¹⁴C]acetate for 48 hr and then washed three times with DMEM and incubated with iAβ at a final concentration of 8 μm. The iAβ-mediated release of cholesterol and PC (b) increased with incubation time. Each data point represents mean ± SE for three samples. Effect of Congo red on iAβ-mediated lipid release and effect of fresh Aβ on lipid release from neurons were investigated using labeled neurons with [¹⁴C]acetate for 48 hr. c, Cells were washed three times with DMEM and then incubated with iAβ (10 μm), iAβ (10 μm) with Congo red (CR) (10 μm), CR alone (10 μm), freshly dissolved Aβ (frAβ) (10 μm), and frAβ plus CR (10 μm) in serum-free N₂ medium for 24 hr. The release of cholesterol and PC in iAβ-treated culture medium was abolished by concurrent treatment with Congo red. Freshly dissolved Aβ1–40 did not promote lipid release from these cells. Each data point represents mean ± SE for four samples. *p < 0.005 versus CONT, iAβ + CR, frAβ, and frAβ + CR. CONT, Control cultures;iAβ, incubated Aβ1–40; CR, Congo red; frAβ, fresh Aβ1–40. d, Cells were washed three times with DMEM and then incubated with none (CONT), iAβ (5 μm), iAβ (5 μm) + NAC (1 mm), NAC (1 mm), H₂O₂ (2 mm), and H₂O₂ (2 mm) + NAC (1 mm). *p < 0.001 versus CONT and NAC; **p < 0.0001 versus H₂O₂ + NAC; ^#p < 0.06 versus CONT and NAC.NAC, N-acetyl-l-cysteine.e, The cultures were washed three times with DMEM and then incubated with none (CONT), iAβ (5 μm), and iAβ (5 μm) + H7 (30 nm) for 16 hr at 37°C, and the lipids in the medium and the cells were quantified as described in Materials and Methods. *p < 0.004 versus CONT and iAβ + H7.

Fig. 3.

Density gradient ultracentrifugation analysis of the culture medium of neurons in the presence of iAβ or H₂O₂. Neuronal cultures plated in six-well plastic plates were incubated with Aβ1–40 (10 μm) in serum-free N₂ medium for 24 hr. The culture medium was collected, filtered through a 0.45 μm filter, and subjected to an initial discontinuous density gradient prepared using KBr solution as described in Materials and Methods. a, After ultracentrifugation, fractions were obtained, and the density, cholesterol, and phospholipids content in each fraction were determined. b, Aliquots of 10 μl from each fraction were mixed with the same volume of SDS buffer, subjected to SDS gel electrophoresis, and immunoblotted with antibodies against Aβ (BA27), apoE (AB947), and apoJ. GM1 ganglioside in each fraction was detected with HRP-conjugated chorea toxin-B. c, Forty-eight hours after plating in serum-free N₂ medium, the neuronal cultures plated in six-well plastic plates were washed in DMEM and incubated with Aβ1–40 (10 μm) or 5 mmH₂O₂ in serum-free N₂ medium for indicated periods. The percentage of LDH released from the cultures was determined as described in Materials and Methods. The data are mean ± SE of triplicate. d, Forty-eight hours after plating in serum-free N₂ medium, the neuronal cultures plated in six-well plastic plates were washed in DMEM and incubated with 5 mm H₂O₂ in serum-free N₂ medium for 24 hr. The culture medium was collected, filtered through a 0.45 μm filter, and subjected to an initial discontinuous density gradient prepared using KBr solution as described in Materials and Methods. After ultracentrifugation, fractions were obtained, and the density and concentrations of cholesterol and phospholipids were determined.

Fig. 4.

iAβ promotes lipid release from astrocytes in culture. Astrocyte-rich cultures were labeled with [¹⁴C]acetate for 48 hr as described in Materials and Methods. Cells were then washed three times with DMEM and incubated with iAβ1–40 or fresh Aβ at various concentrations for 4 hr. The conditioned media were collected and then filtered. The lipids that were released into the medium and the intracellular lipids were extracted and analyzed as described in Materials and Methods. iAβ (○) promoted the release of cholesterol (a) and phosphatidylcholine (PC) (b) in a dose-dependent manner; fresh Aβ (●) did not. Data are mean ± SE for four samples. The scale bars are smaller than the symbol size at 0 μm (a and b). Density gradient ultracentrifugation analysis was performed with the conditioned medium of astrocytes in the presence of iAβ. Astrocytes plated in six-well plastic plates were incubated with Aβ1–40 (10 μm) in DMEM for 24 hr. The culture medium was collected, filtered through a 0.45 μm filter, and subjected to an initial discontinuous density gradient prepared using KBr solution as described in Materials and Methods. After ultracentrifugation, the solution was fractionated. The density and cholesterol and phospholipid content in each fraction were determined (c). Aliquots of 10 μl from each fraction were mixed with the same volume of SDS buffer, subjected to SDS gel electrophoresis, and immunoblotted with antibodies against Aβ (BA27), apoE (AB947), and apoJ. GM1 ganglioside in each fraction was detected with HRP-conjugated chorea toxin-B (d).

Fig. 5.

Electron micrographs of lipid particles associated with iAβ and apoE3. Neuronal cultures were incubated with 10 μm iAβ or 0.25 μm apoE3 for 24 hr. The conditioned media of these cultures were collected, filtered with a 0.45 μm Millipore filter, subjected to an initial discontinuous density gradient prepared using KBr solution, and centrifuged at 34,500 rpm for 48 hr using a SW 41-Ti rotor. After centrifugation, 12 fractions were isolated, and lipid concentration and the density in each fraction were determined. HDL fractions were then diluted with 10-fold volumes of distilled water, followed by centrifugation at 34,500 rpm for 48 hr using a SW 41-Ti rotor. Electron microscopic examination of the lower part of each solution was performed. Negative staining of electron micrographs of lipid particles in the presence of apoE and iAβ is shown (a and b, respectively). Results of immunoelectron microscopy show that exogenously added iAβ1–40 forms complexes with lipid particles as demonstrated using the antibody directed against human Aβ1–17, 6E10 (Kim et al., 1990). c, Gold labeling is considered to be associated with lipid particles. d, In contrast, lipid particles were not labeled with gold without 6E10. Scale bar, 50 nm.

Fig. 6.

Binding of Aβ by immobilized anti-Aβ antibody, 6E10, results in capture of lipids. Filtered conditioned medium of neuronal and astrocyte cultures incubated with iAβ for 48 hr was subjected to immunoprecipitation using anti-Aβ antibody (6E10), anti-apoE antibody (AB947), anti-apoJ antibody, and normal mouse IgG.A, The protein-G-Sepharose-associated lipids were determined using the kits described in Materials and Methods. The quantity of cholesterol (a) and phospholipids (b) immunoprecipitated with 6E10 from the conditioned medium of neuronal culture incubated with iAβ was ∼12% of the total cholesterol and phospholipids in the initial conditioned medium. However, those immunoprecipitated with anti-apoE antibody, anti-apoJ antibody, or normal mouse IgG were significantly low.B, In astrocyte culture medium, the quantity of cholesterol (a) and phospholipids (b) immunoprecipitated with 6E10 and AB947 from the conditioned medium incubated with iAβ was significantly higher than those with anti-apoJ antibody or normal mouse IgG. Western blot analysis using anti-apoE antibody (AB947) and anti-Aβ antibody (BA27) was performed with the immunoprecipitates. The bands corresponding to Aβ monomers and dimers is labeled only in immunoprecipitates by 6E10, whereas no band or a faint band was detected in those by AB947, anti-apoJ antibody, and normal mouse IgG in neurons (Ac). The bands corresponding to Aβ monomers and dimers and apoE are labeled in both immunoprecipitates by 6E10 and AB947, respectively, whereas no band or a faint band was detected in those by anti-apoJ and normal mouse IgG for astrocytes (Bc). Data are mean ± SE for four samples. *p < 0.01 versus 6E10, anti-apoJ, and normal IgG (Ac); *p < 0.01 versus anti-apoJ and normal IgG (Bc).

Fig. 7.

Binding affinity and internalized efficacy of Aβ-lipid particles into neurons. Astrocyte-rich cultures were labeled with [¹⁴C]acetate for 48 hr as described in Materials and Methods. Cells were then washed three times with DMEM and incubated with 10 μm iAβ 1–40 or 0.25 μmhuman recombinant apoE3 for 24 hr. The conditioned media were obtained, filtered using a 0.45 μm filter, and subjected to density-gradient ultracentrifugation at 34,000 rpm for 48 hr in a Beckman SW 41-Ti rotor. The HDL fractions were then collected and dialyzed. The radioactivity in each sample was determined by a scintillation counter and normalized with DMEM. The normalized conditioned medium containing iAβ or apoE was added to neuronal cultures at 4 or 37°C. Twenty minutes after the addition, the cultures were washed three times with cold PBS and dried under air flow at room temperature. The lipids in each culture were extracted by incubation with hexane/isopropanol (3:2 v/v) solution for 1 hr. Then the solution was moved into tubes and dried under N₂ gas. The extracted lipids were then dissolved in chloroform, developed in HPTLC, and quantified by BAS2500 (Fuji Film, Tokyo, Japan). a, The ratio of the labeled cholesterol associated with neurons was significantly lower at both 4 and 37°C in the cultures incubated with conditioned medium treated with iAβ1–40. However, it was significantly higher at both 4 and 37°C in the cultures incubated with apoE. Similar results were observed for the ratio of the labeled phosphatidylcholine in association with the cells (b). Data are mean ± SE for six samples. *p < 0.003.