ApoE promotes the proteolytic degradation of Abeta

Abstract

Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Abeta homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Abeta from the brain. The endolytic degradation of Abeta peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Abeta degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Abeta load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Abeta from the brain and suggest that LXR agonists may represent a novel therapy for AD.

Figure 1. Microglia efficiently take up and degrade soluble Aβ

(A) Kinetics of soluble Aβ uptake by microglia. BV2 microglia were incubated with exogenous Alexa488-labeled soluble Aβ₄₂ for the indicated periods. The uptake of fluorescently-labeled soluble Aβ was measured using flow cytometry. (B) Soluble Aβ is dose-dependently taken up by microglia. BV2 microglia were incubated for 3 hours with indicated concentrations of unmodified soluble Aβ₄₂. The intracellular Aβ₄₂ levels were measured using ELISA. The results were normalized to total cellular protein. (C) Soluble Aβ is rapidly trafficked to lysosomes for degradation. Confocal imaging of live BV-2 microglia 15 min after addition of 2 µg/ml soluble Cy3-Aβ₄₂ demonstrated localization of Aβ (red) within lysosomes. Lysosomes were stained using LysoTracker (green). (D) Exogenous soluble A β was efficiently cleared from the medium by microglia. BV2 microglia were incubated with 2 µg/ml Aβ₄₂ for the indicated time. The media were collected and immunoblotted for Aβ(***P<0.001; ###P<0.001). (E) Soluble Aβ can be degraded by microglia. BV2 microglia were incubated for 3 hours with 2 µg/ml unmodified soluble Aβ₄₂. After washout of the remaining Aβ₄₂ in the media, the intracellular Aβ₄₂ levels were measured using ELISA. The media were also monitored for resecreted Aβ. (F) Neprilysin and related proteinases mediate intracellular degradation of soluble Aβ by microglia. Primary microglia from wild type mice were pretreated with vehicle, 10 µM phosphoramidon or 1 µM thiorphan for 18 hours. The cells were then incubated with 2 µg/ml Aβ₄₂ in the presence of vehicle or drug for an additional 24 hours. The data represent the outcome of 3 independent experiments (*P<0.05).

Figure 2. HDL Apolipoproteins increase soluble Aβ degradation in microglia

BV2 microglia (A) or primary microglia from wild type mice (B) were incubated with 2 µg/ml Aβ₄₂ in the presence or absence of 20 µg/ml ApoA-I or 5 µg/ml ApoE for 3 hours or 24 hours respectively. The intracellular Aβ₄₂ levels were quantified from three independent experiments and data normalized to loading controls. Values are expressed as percentage relative to the value of controls (**P<0.01; ***P<0.001). (C) ApoE dose-dependently increased Aβ degradation in microglia. BV2 microglia were incubated with 2 µg/ml Aβ₄₂ in the presence of increasing concentrations of purified human ApoE (0, 0.2, 1 or 5 µg/ml) for 3 hours, intracellular Aβ₄₂ levels were quantified using ELISA and data normalized to total protein (*P<0.05). (D–F) ApoE enhanced soluble Aβ degradation. BV2 microglia were incubated with 2 µg/ml Aβ₄₂ or 2 µg/ml Alexa488-labeled Aβ₄₂ in the presence or absence of 1 µg/ml purified human ApoE for indicated time. (D) The remaining intracellular Aβ₄₂ levels were quantified using ELISA and data normalized to total protein (***P<0.001). (E) The total internalized Aβ₄₂ over the same intervals was monitored using flow cytometry. (F) The efficiency of Aβ degradation was expressed as a ratio of remaining intact Aβ₄₂ over total internalized Aβ₄₂ (***P<0.001).

Figure 3. LXR activation enhances intracellular Aβ degradation in microglia

BV2 microglia (A) or primary microglia from wild type mice (B) were pretreated with DMSO or 1 µM GW3965 for 18 hours. The cells were then incubated with 2 µg/ml Aβ₄₂ in the presence or absence of 20 µg/ml ApoA-I for 3 hours or 24 hours, respectively. Cellular lysates were subjected to SDS-PAGE and Western blotted for ABCA1, ApoE, Aβ and β-actin or β-tubulin. The levels of Aβ were normalized to β-actin or β-tubulin as loading controls. Relative intracellular Aβ levels were quantified from three independent experiments (**P<0.01; ***P<0.001; ##P<0.01). (C) Primary microglia from wild type mice were pretreated with DMSO, 1 µM GW3965 or 1 µM T0901317 for 18 hours. The cells were then incubated with 2 µg/ml Aβ₄₂ in the presence or absence of 5 µg/ml purified human plasma ApoE for 24 hours. Intracellular Aβ levels were quantified using ELISA for Aβ₄₂ and normalized to total protein. The data represent the outcome of 4 independent experiments (***P<0.001; #P<0.05; ###P<0.001).

Figure 4. ApoE is essential for efficient degradation of Aβ

(A) Loss of ApoE resulted in intracellular accumulation of Aβ. Primary microglia from wild type or Apoe −/− mice were treated with 2 µg/ml Aβ₄₂ for 24 hours. The lysates were subjected to SDS-PAGE and Western blotted for ABCA1, ApoE, Aβ and β-tubulin as a loading control. Blots shown are representative of three independent experiments. (B) Intracellular Aβ levels were quantified using ELISA for Aβ₄₂ and normalized to total protein. The data represent the outcome of 5 independent experiments (**P<0.01). (C) Exogenous ApoE rescued the Aβ degrading deficiency in Apoe −/− microglia. Primary microglia from Apoe −/− mice were incubated with 2 µg/ml Aβ₄₂ in the absence or presence of 1 µg/ml ApoE for an additional 24 hours. Intracellular Aβ₄₂ levels were quantified using ELISA and the data normalized to total protein (***P<0.001). (D) ApoE is required for LXR-mediated effect on Aβ degradation. Primary microglia from Apoe −/− mice were pretreated with DMSO or 1 µM GW3965 for 18 hours. The cells were then incubated with 2 µg/ml Aβ₄₂ for an additional 24 hours. Intracellular Aβ₄₂ levels were quantified using ELISA and data normalized to total protein. (E) Native ApoE particles enhanced Aβ degradation in an isoform-dependent manner. Primary microglia from ApoE −/− mice were treated with 2 µg/ml Aβ₄₂ in the absence or the presence of 200 ng/ml purified ApoE-containing native HDL particles isolated from immortalized astrocytes expressing the human ApoE isoforms for 24 hours. The levels of remaining intracellular Aβ were quantified using ELISA for Aβ₄₂. The data represent the outcome of 4 independent experiments (**P<0.01; ***P<0.001; #P<0.05).

Figure 5. ABCA1 influences the intracellular degradation of Aβ by microglia

(A) Loss of Abca1 impairs Aβ degradation in primary microglia. Primary microglia from Abca1 +/+, +/− and −/− mice were treated with 2 µg/ml Aβ₄₂ for 24 hours. The cellular levels of Aβ₄₂ were measured in lysates by ELISA. The data were normalized to total protein and represent the outcome of 5 independent experiments. (B) Primary microglia from Abca1 +/+ and −/− mice were pretreated with DMSO or 1 µM GW3965 for 18 hours. The cells were then incubated with 2 µg/ml Aβ₄₂ in the presence or absence of 1 µM GW3965 for 24 hours. Cellular lysates were subjected to SDS-PAGE and Western blotted for ABCA1, ApoE, Aβ and β-actin. Representative blots from 3 independent experiments are shown. (C) Primary microglia from Abca1 +/+ and −/− mice were treated as above. Intracellular Aβ levels were quantified using ELISA for Aβ₄₂ and normalized to total protein. The data represent the outcome of 6 independent experiments (*P<0.05; ***P<0.001).

Figure 6. Extracellular degradation of soluble Aβ is dependent on IDE and related proteinases, and is influenced by the lipidation status of ApoE

(A) Astrocyte-conditioned medium degrades soluble Aβ by IDE and related proteinases. Astrocyte-conditioned medium from wild type mice was incubated with Aβ₄₂ in the presence or absence of 10 µM insulin for 24 hours. The samples were then subjected to SDS-PAGE and Western blotted for ApoE and Aβ. (B) After 24 hours, the samples were analyzed for Aβ₄₂ using ELISA. The data represent the outcome of 3 independent experiments (**P<0.01). (C) Lipidation status of ApoE regulates Aβ degradation in astrocyte-conditioned medium. Conditioned media from astrocytes derived from Abca1 +/+, +/−, and −/− mice were incubated with 1 µg/ml Aβ₄₂ for 0 or 24 hours and the reaction mixtures were subjected to SDS-PAGE and Western blotted for ApoE and Aβ. Representative blots from three independent experiments are shown. (D) The amount of Aβ₄₂ remaining in the medium after 24 hours was quantified using ELISA. The data represent the outcome of 3 independent experiments (**P<0.01; ##P<0.01). (E) ApoE-containing HDL particles promote Aβ degradation by recombinant IDE. ApoE-containing HDL particles were collected from astrocyte-conditioned medium derived from wild type, Abca1−/− and Apoe−/− mice by immunoprecipitation and the complexes were incubated with 500 ng/ml recombinant IDE and 2 µg/ml Aβ₄₂ for 1 hour. The reaction mixtures were then resolved by SDS-PAGE and Western blotted for IDE, ApoE and Aβ.

Figure 7. LXR agonist treatment reduces Aβ levels and ameliorates plaque burden in Tg2576 mice

(A) Aged Tg2576 mice (12 month-old) or genetically similar controls were treated for 4 months with normal chow or chow containing GW3965 (120 mg/kg, 33 mg/kg/day). Aβ plaque burden was monitored by 6E10 staining in the hippocampus. Plaque number and plaque area were quantified in (B) and (C) respectively (n=5, *P<0.05; **P<0.01). The levels of Aβ₄₀ (D) and Aβ₄₂ (E) were quantified using ELISA (*P<0.05). (F) The levels of full-length APP, C99 C-terminal fragment, total Aβ, ABCA1 and ApoE were monitored by Western analysis. The results were normalized to β-actin (n=5, *P<0.05; **P<0.01). Quantification of the data is shown under the Western blots.

Figure 8. GW3965 significantly improves contextual memory in Tg2576 mice

Tg2576 or wild type mice (20 week-old) were orally treated with vehicle or 50 mg/kg/day of GW3965 for 6 days and subjected to contextual memory assessment as outlined in the methods. Treatment of the LXR agonist improved hippocampal-dependent contextual memory in the heterozygous Tg2576 mice (n = 11, *P<0.05). No significant effect of treatment was observed in contextual memory in the wild-type littermate control mice. No significant effect of genotype was observed in hippocampal-independent cue conditioning (data not shown).

Figure 9. ApoE and its lipidation status regulates the proteolytic degradation of soluble Aβ

The level of soluble Aβ is homeostatically controlled by its production by neurons and its subsequent clearance. Soluble Aβ can be cleared by proteolytic enzymes including NEP and IDE acting both intracellularly and extracellularly. ApoE is principally synthesized and secreted by glia. The lipid transporter ABCA1 mediates the lipidation of ApoE. Liver X receptors regulate the expression of both ABCA1 and ApoE and their activation results in increased levels of lipidated ApoE. The degradation of soluble Aβ both intracellularly and extracellularly is enhanced by increasing ApoE and its lipidation.