Blocking the apolipoprotein E/amyloid-beta interaction as a potential therapeutic approach for Alzheimer's disease

Abstract

The amyloid-beta (Abeta) cascade hypothesis of Alzheimer's disease (AD) maintains that accumulation of Abeta peptide constitutes a critical event in the early disease pathogenesis. The direct binding between Abeta and apolipoprotein E (apoE) is an important factor implicated in both Abeta clearance and its deposition in the brain's parenchyma and the walls of meningoencephalic vessels as cerebral amyloid angiopathy. With the aim of testing the effect of blocking the apoE/Abeta interaction in vivo as a potential novel therapeutic target for AD pharmacotherapy, we have developed Abeta12-28P, which is a blood-brain-barrier-permeable nontoxic, and nonfibrillogenic synthetic peptide homologous to the apoE binding site on the full-length Abeta. Abeta12-28P binds with high affinity to apoE, preventing its binding to Abeta, but has no direct effect on Abeta aggregation. Abeta12-28P shows a strong pharmacological effect in vivo. Its systemic administration resulted in a significant reduction of Abeta plaques and cerebral amyloid angiopathy burden and a reduction of the total brain level of Abeta in two AD transgenic mice models. The treatment did not affect the levels of soluble Abeta fraction or Abeta oligomers, indicating that inhibition of the apoE/Abeta interaction in vivo has a net effect of increasing Abeta clearance over deposition and at the same time does not create conditions favoring formation of toxic oligomers. Furthermore, behavioral studies demonstrated that treatment with Abeta12-28P prevents a memory deficit in transgenic animals. These findings provide evidence of another therapeutic approach for AD.

Fig. 1.

Aβ12-28P binds to apoE and abolishes its effect on Aβ fibrillization. (a) Shown is a solid-phase binding assay of lipidated human apoE4 isoform to Aβ1-40, Aβ12-28P, and Aβ12-28 synthesized from l-amino acids. K_D values represent mean ± SEM from three independent experiments. (b) Shown is dose-dependent inhibition of the apoE4/Aβ binding by increasing concentrations of Aβ12-28. P values represent mean ± SEM from three independent experiments. (c) Thioflavin-T aggregation assay demonstrates the effect of Aβ12-28P on the apoE/Aβ interaction. Adding the human lipidated apoE4 complexes dramatically increases the amount of Aβ1-40 fibrils formed over time (P = 0.007, repeated measures ANOVA; P < 0.01 for specific post hoc comparison of Aβ1-40 + apoE4 versus Aβ1-40 alone). Preincubation of apoE with Aβ12-28P abolishes the apoE effect on Aβ1-40 aggregation (P < 0.01 and nonsignificant post hoc analysis for the specific effect of Aβ+apoE/Aβ12-28P versus Aβ + apoE and Aβ, respectively). Aβ12-28P alone has no effect on the aggregation of Aβ1-40 (nonsignificant). Aβ12-28P does not aggregate over time. (d) Shown is a lack of direct effect of Aβ12-28P on Aβ1-40 aggregation. Aβ1-40 (200 μmol/liter) was incubated in the presence of Aβ12-28P concentrations ranging from 0 to 200 μmol/liter (repeated measures ANOVA P = 0.573).

Fig. 2.

Treatment with Aβ12-28P rescues APP_K670N/M671L mice from memory decline. Aβ12-28P-treated APP_K670N/M671L mice performed comparably to WT, age- and sex-matched littermates on radial arm maze testing. Both groups performed statistically better than APP_K670N/M671L mice treated with vehicle; ANOVA P < 0.0001, post hoc Aβ12-28P vs. WT nonsignificant, Aβ12-28P vs. vehicle P < 0.001, WT vs. vehicle P < 0.001 (n = 11 for vehicle and Aβ12-28P-treated Tg groups, n = 12 for WT).

Fig. 3.

Treatment with Aβ12-28P reduces Aβ deposition in APP_K670N/M671L and APP_K670N/M671L/PS1_M146L mice. (a) Decrease in the total Aβ burden in the neocortex, the cingulated cortex, and the hippocampus as quantified by unbiased hierarchical sampling (n = 11; ∗, P < 0.01; ∗∗, P < 0.001). (b) Hemispheric sections from 7-month-old APP_K670N/M671L/PS1_M146L mice treated with vehicle (Left) and Aβ12-28P (Right) depict the difference in Aβ burden. Immunostaining was done with a mixture of 4G8 and 6E10 anti-Aβ mAbs. (c) Shown is a reduction in the fibrillar Aβ burden in Aβ12-28P-treated animals (n = 11; ∗, P < 0.05). (d) Shown is the cingulate cortex and the neocortex of 18-month-old vehicle-treated (Left) and Aβ12-28P-treated (Right) APP_K670N/M671L mice stained with Thioflavin-S. There is a clearly visible reduction in the burden of parenchymal Aβ deposits (yellow arrowhead) and CAA (white arrowhead). Both types of deposits were quantified separately. (e) Shown is a decrease in the CAA burden in Aβ12-28P-treated APP_K670N/M671L mice (∗, P < 0.05). (f) Shown is Perls staining of parenchymal vessels in Aβ12-28P-treated APP_K670N/M671L mice, revealing a lack of microhemorrhages.

Fig. 4.

Treatment with Aβ12-28P reduces the amount of apoE present in Aβ deposits. (a) Shown is double immunofluorescent staining colocalizating apoE in Aβ deposits in the hippocampus of APP_K670N/M671L mice. Only a minority of plaques were apoE-negative in both groups (see white arrowheads). (b) Shown is a reduction in the burden of apoE-positive deposits in Aβ12-28P-treated animals (∗, P < 0.01; ∗∗, P < 0.001). Values are averaged for all three areas of interest. (c) Shown is the reduction in the mean optic density (O.D.) index of apoE deposits in Aβ12-28P-treated animals (∗, P < 0.05).

Fig. 5.

Treatment with Aβ12-28P decreases the levels of total Aβ40 and Aβ42 but does not alter levels of the soluble Aβ fraction or Aβ oligomers. (a) Shown is a statistically significant decrease in the levels of FA-extracted (FA_extr) Aβ40 and Aβ42 (total Aβ) in APP_K670N/M671L mice (Left) and APP_K670N/M671L/PS1_M146L mice (Right). The level of soluble Aβ40 and Aβ42 fractions extracted with DEA (DEA_extr) did not differ between groups (n = 11; ∗, P < 0.05; ns, not significant). (b) A Western blot of brain homogenates stained with A11 oligomer-specific polyclonal antibody. The density and thickness of oligomer bands did not differ between vehicle-and Aβ12-28P-treated 18-month-old APP_K670N/M671L mice. No oligomers were detected in age-matched WT littermates. (c) The densitometric analysis of oligomer-specific bands. There is no significant difference between Aβ12-28P- and vehicle-treated groups in either Tg model.