ApoE influences amyloid-β (Aβ) clearance despite minimal apoE/Aβ association in physiological conditions

Abstract

Apolipoprotein E gene (APOE) alleles may shift the onset of Alzheimer's disease (AD) through apoE protein isoforms changing the probability of amyloid-β (Aβ) accumulation. It has been proposed that differential physical interactions of apoE isoforms with soluble Aβ (sAβ) in brain fluids influence the metabolism of Aβ, providing a mechanism to account for how APOE influences AD risk. In contrast, we provide clear evidence that apoE and sAβ interactions occur minimally in solution and in the cerebrospinal fluid of human subjects, producing apoE3 and apoE4 isoforms as assessed by multiple biochemical and analytical techniques. Despite minimal extracellular interactions with sAβ in fluid, we find that apoE isoforms regulate the metabolism of sAβ by astrocytes and in the interstitial fluid of mice that received apoE infusions during brain Aβ microdialysis. We find that a significant portion of apoE and sAβ compete for the low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cellular uptake pathway in astrocytes, providing a mechanism to account for apoE's regulation of sAβ metabolism despite minimal evidence of direct interactions in extracellular fluids. We propose that apoE influences sAβ metabolism not through direct binding to sAβ in solution but through its actions with other interacting receptors/transporters and cell surfaces. These results provide an alternative frame work for the mechanistic explanations on how apoE isoforms influence the risk of AD pathogenesis.

Keywords: cholesterol efflux; neurodegeneration.

Fig. 1.

Characterization of rapoE isoforms and cell-secreted sAβ. (A) H4 APP695∆NL cells were incubated with serum-free Opti-MEM medium for 24 h. Then the medium was collected and concentrated fivefold with a 3-kDa cutoff concentrator and subjected to SEC. The SEC-purified Aβ was separated in a 16.5% tricine gel with nondenaturing PAGE and immunoblotted with 82E1 (anti–Aβ1–5) antibody (Right). (B) rapoE2, rapoE3, and rapoE4 particles (8 µg) were loaded on a 4–20% Tris⋅glycine gel for native PAGE to assess particle size using a standard containing proteins of specified hydrodynamic radii; particles that were concentrated for in vivo microdialysis experiments in Fig. 6 also were analyzed by native PAGE. (C) [³H]-cholesterol–labeled H4 APP695∆NL cells were incubated with 20 µg rapoE3 (1:50:10) and rapoE4 (1:50:10) particles at different time points to assess cholesterol efflux relative to liposomes, expressed as the percentage of radiolabeled cholesterol released into the medium. Differences were assessed using Student's t tests (n = 4; P < 0.05).

Fig. 2.

KBr density gradient studies showing that the association of apoE with cell-secreted sAβ in the presence and absence of cells is minimal. (A) H4 APP695∆NL cells were incubated with 20 μg of lipidated rapoE3 particles and liposome particles. Medium was collected at indicated time points and subjected to ultracentrifugation in a KBr density gradient. Aliquots of the density gradient were used to determine radioactivity and density. rapoE3 (1:50:10) particles were distributed primarily below the density of 1.15 g/cm³. (B) H4 APP695∆NL cells were incubated with 20 μg of lipidated rapoE3 particles (1:50:10) and liposomes (phospholipid:cholesterol ratio, 50:10) for 3, 6, and 12 h [Aβ:apoE molar ratios of 1:50 (3 h), 1:35 (6 h), and 1:25 (12 h)]. Medium was collected at indicated time points and subjected to ultracentrifugation in a KBr density gradient. Aliquots of the density gradient were used for Aβ_1-x ELlSA. (C) (Left) The bar graph represents the percentage of sAβ present in the region with a KBr density less than 1.15 g/cm³ (percent Aβ bound) and in the region with a KBr density greater than 1.15 g/cm³ (percent Aβ unbound). (Right) Time-dependent binding of Aβ and rapoE3. Differences were assessed using one-way ANOVA followed by a Dunnet post test (n = 5). (D) The bar graph represents the percentage of sAβ present in the region with a KBr density less than 1.15 g/cm³ (percent Aβ bound) and in the region with a KBr density greater than 1.15 g/cm³ (percent Aβ unbound) when H4 APP695∆NL cells were incubated with 20 μg of lipidated rapoE2, rapoE3, rapoE4 (1:50:10) particles and mouse astrocyte-derived apoE particles for 12 h (Aβ:apoE molar ratio, 1:25). Differences were assessed using Student's t tests (n = 5). (E) The bar graph represents the percentage of sAβ present in the region with a KBr density less than 1.15 g/cm³ (percent Aβ bound) and in the region with a KBr density greater than 1.15 g/cm³ (percent Aβ unbound) when H4 APP695∆NL CM-derived Aβ was incubated with 20 μg of lipidated rapoE2, rapoE3, rapoE4 particles (in a ratio of 1:50:10) for 12 h (Aβ:apoE molar ratio, 1:25 at 12 h) without cells. Differences were assessed using one-way ANOVA followed by a Dunnet posttest (n = 4).

Fig. 3.

SEC studies showing that the association of apoE with cell-secreted sAβ is minimal. (A and B) H4 APP695∆NL CM-derived sAβ (100 ng/mL) was incubated with 10 μg of astrocyte-derived apoE3 and apoE4 particles for 6 h (A) or 20 μg of rapoE3 and rapoE4 particles (1:50:10) for 3 or 6 h (B). Medium was subjected to SEC with a Superose 6/10 column. Fractions were analyzed for apoE and Aβ by ELISAs. (C) The bar graph represents the percentage of sAβ present in the region of apoE elution, detected by ELISA (fraction number, 25–32) (percent Aβ bound) and in the region in which apoE is not detectable with ELISA (fraction number, 34–40) (percent Aβ unbound). Differences were assessed using t tests (n = 5). (D) [³H]-cholesterol–labeled H4 cells were incubated with 20 µg rapoE3 particles (1:50:10) and the indicated concentrations of Aβ purified from H4 APP695∆NL CM at different time points to assess cholesterol efflux, expressed as the percentage of radiolabeled cholesterol released into the medium. The efflux properties of apoE3 did not change significantly in the presence or absence of Aβ. Significance was assessed using one-way ANOVA followed by a Dunnet posttest (n = 4).

Fig. 4.

FCS study showing that the association of apoE with synthetic Aβ is negligible. (A and B) Diffusion time was obtained from incubation of 100 nM TMR, 100 nM TMR-Aβ40 and TMR-Aβ42 and 100 nM rapoE2, rapoE3, and rapoE4 particles (molar ratio 1:1) for 2 h or 24 h (n = 4). (C) Diffusion time was obtained from incubation of 100 nM and 500 nM TMR-Aβ42 and 100 nM astrocyte-derived apoE2, apoE3, and apoE4 for 24 h (n = 4).

Fig. 5.

Association of apoE with sAβ in human APOE ε3/ε3 and APOE ε4/ε4 CSF is minimal. (A) CSF from APOE ε3/ε3 and APOE ε4/ε4 human subjects (0.5 mL) was subjected to SEC. Fractions were analyzed for apoE and Aβ_1-x by ELISAs as described in SI Materials and Methods. Differences were assessed using Student's t tests (n = 4). (B) Pooled CSF from human subjects (0.8 mL) was incubated with Aβ purified from CM of H4 APP695∆NL cells for 6 h and subjected to SEC. Fractions were analyzed for apoE and Aβ_1-x by ELISAs as described in SI Materials and Methods. Differences were assessed using Student's t tests (n = 4).

Fig. 6.

Aβ and apoE share clearance pathways without direct binding. (A and B) To assess the effect of apoE isoforms on Aβ uptake, apoE2, apoE3, apoE4, and apoE-KO astrocytes were incubated with CM from H4 APP695∆NL cells secreting sAβ (sAβ, 100 ng/mL) for 12 h. The amount of Aβ in the cell lysate (A) and medium (B) then was assessed by ELISA as described in Materials and Methods. (C) apoE2, apoE3, apoE4, and apoE-KO–expressing astrocytes were incubated with CM from 7PA2 cells (sAβ,100 ng/mL) for 12 h. The amount of Aβ in the cell lysate then was assessed by ELISA as described in SI Materials and Methods. (D–F) To determine whether apoE and Aβ compete for the same cellular uptake pathways, apoE-KO astrocytes were incubated with CM from H4 APP695∆NL cells (sAβ,100 ng/mL) and indicated concentrations of rapoE2, rapoE3, and rapoE4 (apoE:phospholipid:cholesterol ratio, 1:50:10) for 12 h. The amount of Aβ in the cell lysate (D) and in the medium from D (E) then was assessed by ELISA as described in SI Materials and Methods. To determine the role of LRP1 in apoE-dependent Aβ competition, MEF1 (LRP1-expressing cells), and MEF2 (LRP1-KO cells) were incubated with CM from H4 APP695∆NL cells (sAβ,100 ng/mL) and 10⁻⁷ M rapoE2, rapoE3, and rapoE4 (apoE:phospholipid:cholesterol ratio, 1:50:10) for 12 h. (F) The amount of Aβ in the cell lysate then was assessed by ELISA as described in SI Materials and Methods. (G) ApoE-KO astrocytes were incubated with CM from H4 APP695∆NL cells (sAβ,100 ng/mL) in the presence or absence of nonimmune IgG or anti-LRP1 IgG (75 µg/mL) and 10⁻⁷ M rapoE3 and rapoE4 (apoE:phospholipid:cholesterol ratio, 1:50:10) for 12 h. The amount of Aβ in the cell lysate then was assessed by ELISA as described in SI Materials and Methods. Significance was assessed using one-way ANOVA followed by a Dunnett posttest (n = 4–6). Data are shown as mean ± SEM (n ≥ 4) *^{, #, +}P < 0.05; ^{**, ##, ++}P < 0.005; ^{***, ###, +++}P < 0.0005; n.s., not significant.

Fig. 7.

Infusing CNS fluid with rapoE particles increases ISF Aβ levels. (A) A 38-kDa cutoff microdialysis probe with a specialized side infusion port was implanted in hippocampi of young PDAPP/TRE mice. After a stable 6-h baseline, 1 μg of freshly prepared rapoE2 or rapoE4 (in artificial CSF solution) was infused at a flow rate of 0.07 μL/min directly at the site of microdialysis in PDAPP/E2 or PDAPP/E4 mice, respectively, and ISF [eAβ_1-x] was monitored for an additional 6 h. Shown is the relative change in [eAβ_1-x] within each mouse after rapoE2 or rapoE4 infusion compared with its mean baseline period. (B) The mean percent change in [eAβ_1-x] following rapoE infusion relative to the baseline period was calculated for each mouse in each group. Differences between baseline and treatment periods were assessed for each group using paired t tests. n = 4–7 mice per group; **P < 0.01.