The binding of apolipoprotein E to oligomers and fibrils of amyloid-β alters the kinetics of amyloid aggregation

Abstract

Deposition of amyloid-β (Aβ) in Alzheimer's disease (AD) is strongly correlated with the APOE genotype. However, the role of apolipoprotein E (apoE) in Aβ aggregation has remained unclear. Here we have used different apoE preparations, such as recombinant protein or protein isolated from cultured astrocytes, to examine the effect of apoE on the aggregation of both Aβ1-40 and Aβ1-42. The kinetics of aggregation, measured by the loss of fluorescence of tetramethylrhodamine-labeled Aβ, is shown to be dramatically slowed by the presence of substoichiometric concentrations of apoE. Using these concentrations, we conclude that apoE binds primarily to and affects the growth of oligomers that lead to the nuclei required for fibril growth. At higher apoE concentrations, the protein also binds to Aβ fibrils, resulting in fibril stabilization and a slower rate of fibril growth. The aggregation of Aβ1-40 is dependent on the apoE isoform, being the most dramatic for apoE4 and less so for apoE3 and apoE2. Our results indicate that the detrimental role of apoE4 in AD could be related to apoE-induced stabilization of the soluble but cytotoxic oligomeric forms and intermediates of Aβ, as well as fibril stabilization.

Figure 1

Time course of TMR-Aβ aggregation in the presence of various forms of the apoE proteins. Panels A–C show the aggregation of TMR-Aβ_1–40: (A) 4 μM TMR-Aβ_1–40 in the presence of 0, 50, 100, and 200 nM recombinant lipid-free apoE4 and (B and C) 4 μM TMR-Aβ_1–40 in the absence or presence of 150 nM recombinant lipid-free apoE (B) and DMPC–apoE (C), respectively. Panels D–F show the aggregation of TMR-Aβ_1–42: 2 μM TMR-Aβ_1–42 in the absence or presence of recombinant lipid-free apoE (50 nM), DMPC–apoE (50 nM), and astrocyte-derived apoE (11 nM) respectively.

Figure 2

Effect of apoE on the Aβ growth phase. The black line represents the time course of aggregation of TMR-Aβ_1–40 (4 μM) in the absence of apoE. The dark gray and light gray lines represent the time courses when 200 nM recombinant lipid-free apoE4 is added during the growth phase at times t₁ and t₂, respectively.

Figure 3

Electron microscopy of the apoE–Aβ complexes. Representative images of the apoE4–Aβ_1–42 solution collected (A) at time zero, (B) during the intermediate phase, and (C) during the growth phase of aggregation. The black dots represent gold nanoparticles attached to the molecules of apoE4 via a six-His tag at the N-terminus of apoE.

Figure 4

Stability of the fibrils. Electron microscopy images following sonication of Aβ_1–42 fibrils that have been incubated in the absence (A) or presence (B) of apoE4. (C) Fluorescence of TMR-Aβ_1–42 following dilution into 2 M urea of fibrillized TMR-Aβ_1–42 that had been previously incubated without (black line) or with apoE4 (gray line) at a 10:1 (Aβ:apoE) molar ratio.

Figure 5

Effect of apoE on Aβ aggregation. The top panel shows Aβ monomers self-assemble to form small oligomers (O1) that grow to larger oligomers (O2) by monomer addition and/or by clustering. The fibrils (F1) are formed within the large oligomers following nucleation. The fibrils subsequently undergo fragmentation (F1 → F2) and growth (F2 → F3). The bottom panel shows that at low concentrations apoE interacts with the Aβ oligomers (O1 and O2) only, but at higher concentrations, it binds to the fibrils (F1–F3), as well. The broken lines indicate slow growth (L1) and nucleation (L2) of the Aβ oligomers and a reduced rate of fragmentation (L3) of the Aβ fibrils due to interactions with apoE. Stabilization of the oligomers increases the duration of the intermediate phase, while stabilization of the fibrils slows the growth phase.