Immunization with Small Amyloid-β-derived Cyclopeptide Conjugates Diminishes Amyloid-β-Induced Neurodegeneration in Mice

Abstract

Background: Soluble oligomeric (misfolded) species of amyloid-β (Aβ) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against Aβ is a promising disease modifying strategy. However, eliciting an immune response against Aβ in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric Aβ, inducing a specific antibody response.

Objective: Here we investigate the protective effects of the experimental vaccine against oligomeric Aβ1-42-induced neuronal fiber loss in vivo.

Methods: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric Aβ1-42 into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled Aβ1-42 peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain.

Results: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R2 = 0.29, p = 0.02).

Conclusion: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms.

Keywords: Alzheimer’s disease; amyloid-β protein (1–42); cholinergic fibers; cyclopeptides; immunization; mice; nucleus basalis of Meynert; stereotactic injection.

Fig.1

Experimental overview. Mice were randomly assigned to be mock-immunized (mock) or immunized. All mice received three rounds of subcutaneous injections in the groin with a 3-week interval. Mock mice received mock vaccine, immunized mice received our trivalent vaccine. Blood samples were collected from the tail vein and the sera was analyzed by ELISA. Based on the anti-oligomeric Aβ_1-42 endpoint titers obtained (ELISA 1), eight well-responding immunized mice and three mock mice were selected for oligomeric Aβ_1-42-induced NBM lesion surgery (group A). Remaining mice received a fourth immunization and another tail puncture. Based on the titers obtained (ELISA 2),eight immunized and four mock mice were selected for NBM lesion surgery (group B). Remaining mice (group C: eight immunized, two mock mice) received a fifth immunization before NBM lesion surgery. Mice from each group were transcardially perfused ten days after NBM lesion surgery. Final blood samples were collected from the heart just before perfusion and sera obtained were analyzed later (ELISA 3).Experimental days are indicated on the left.

Fig.2

Average of the ¹⁰log value of anti-oligomeric Aβ_1-42 ELISA endpoint titers of immunized and mock mice before and after Aβ_1-42-induced NBM lesions. After all mice had received three (mock-) immunizations, mice with high titers (ELISA 1) and randomly selected mock mice were selected as the first group to receive NBM lesion surgery (group A). Remaining mice received a fourth (mock-) immunization. Again, mice with high titers (ELISA 2) and randomly selected mock mice were selected as the second group to receive NBM lesion surgery (group B). Remaining mice (group C) received a fifth (mock-) immunization before lesion surgery. Measurements before NBM lesions (ELISA 1 and 2) were performed on sera from blood samples collected from the tail vein. Measurements after NBM lesions (ELISA 3) were performed on sera from blood samples taken from the heart, just before transcardial perfusion. The results show that three rounds of immunizations are highly effective, while a fourth and fifth round of immunizations have only minor added effect. Vertical axis categories are numbered between square brackets for easy reference. Error bars represent SEM. Statistical indicators: ^*p < 0.05, ^***p < 0.001, ns indicates p > 0.05.

Fig.3

Active immunization prevents the loss of cholinergic innervations induced by oligomeric Aβ_1-42 injected into the NBM. Shown are representative images of ChAT-positive fibers in the parietal neocortex of mock (A and B) and immunized mice (C and D). Images on the left (A and C) show the side of the brain injected with scrambled Aβ_1-42 control peptide (Aβ-scr). Images on the right (B and D) show the contralateral lesion-sides of the brain injected with oligomeric Aβ_1-42 (Aβ-oligo). Note that in practice the compounds were randomly injected in the left- or right side of the brain. Comparing control and lesion sides of the brain, immunized mice show reduced fiber loss compared to mock mice. In each image the area between the parallel bars indicate the quantified area (layer V of the somatosensory cortex). The horizontal scale bar shown in (D) applies to all images and represents 100 μm.

Fig.4

Effectiveness of the oligomeric Aβ_1-42-induced NBM lesion model and effects in mock and immunized mice. All mice consistently showed less cortical cholinergic fiber density on the Aβ injected side of the brain, as compared to the scrambled-Aβ (Aβscr) injected control side of the brain (A). Compared to mock mice, immunized mice showed significantly reduced cholinergic fiber loss in the cortex, as measured by optical fiber density after ChAT immunostaining (B). In both panels the error bars represent SEM.

Fig.5

Correlation of the ¹⁰log value of anti-oligomeric Aβ_1-42 endpoint titers (x-axis) versus cholinergic fiber loss (y-axis). Titers were obtained by ELISA of sera obtained from blood samples taken from the tail vein on day 54 (ELISA 1), after which all mice had received three immunization injections. Percentage of fiber loss was calculated for each animal based on ChAT-stained cholinergic fiber density in the bilateral parietal neocortex, which is the target of afferent cholinergic pathways from the bilateral NBM sites. The Aβ_1-42 injected lesion side of the brain was compared to the scrambled-Aβ_1-42 injected control side of the brain to determine the percentage of cholinergic fiber loss. The linear regression line and Pearson correlation is based on the immunized mice (indicated by black circles) including the non-responders (separately indicated by open triangles), but not including the mock mice (indicated by grey circles). The R-squared and two-tailed p-value is indicated in the figure.