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

Background: Soluble oligomeric (misfolded) species of amyloid-beta (A beta) 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 A beta is a promising disease modifying strategy. However, eliciting an immune response against A beta 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 A beta, inducing a specific antibody response. Objective: Here we investigate the protective effects of the experimental vaccine against oligomeric A beta(1-42)-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 A beta(1-42) into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled A beta(1-42) 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 (R-2 = 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

A Cyclic Undecamer Peptide Mimics a Turn in Folded Alzheimer Amyloid β and Elicits Antibodies against Oligomeric and Fibrillar Amyloid and Plaques

The 39- to 42-residue amyloid β (Aβ) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aβ immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aβ, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aβ-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aβ(22–28)-YNGK′ elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound—although more weakly—to Aβ(1–42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aβ deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice

IgG response of individual Balb/c mice against Aβ (1–42) as determined by ELISA.

<p>As indicated on the x-axis, groups of eight mice were immunized with cyclo[Aβ (22–28)-YNGK′]-TTd, oligomeric Aβ(1–42), or fibrillar Aβ (1–42). <b>A</b>, coating antigen: oligomeric Aβ (1–42); <b>B</b>, coating antigen: fibrillar Aβ (1–42). A mouse with a serum titer ≤100 is considered to be a non-responder. The two titers out of range in the right panel of <b>A</b> have values of 40<b>×</b>10³ and 50<b>×</b>10³. The figure demonstrates that anti- cyclo[Aβ (22–28)-YNGK′] antibodies recognize oligomeric and fibrillar Aβ (1–42).</p

Western blot of a lysate of SW13 adrenal cells transfected with amyloid precursor protein (APP).

<p>The cells were transfected with either human APP⁶⁹⁵wt (wild type) or with APP⁶⁹⁵swe (Swedish mutation). Monoclonal antibody 6E10 is specific for the largely unstructured N-terminus of amyloid β (Aβ) and recognized both APPs (amino acid residues 2–8 in Aβ correspond to residues 598–604 in APP⁶⁹⁵). Anti-cyclo[Aβ (22–28)-YNGK′] antibodies stained neither of the APPs under the denaturing conditions of the assay and thus seem to recognize a conformational epitope in Aβ.</p

Early folding of human amyloid β (Aβ), around S26 and N27.

<p>The figure is a simplification of the model designed by Olofsson <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019110#pone.0019110-Olofsson1" target="_blank">[28]</a>. Residues in red are solvent accessible; residues in blue (and to a lesser extent in gray) are shielded from the solvent. The peptide cyclo[Aβ(22–28)-YNGK′] is a mimic for misfolded Aβ. YNGK′ is a turn-stabilizing sequence and K′ is a side-chain-modified lysyl residue for selective conjugation to a protein carrier. Other YNGK′-containing cyclic peptides prepared (23–28, 24–29, 25–30, 21–27, 22–28, 23–29, 24–30, and 25–31) did not mimic misfolded Aβ.</p

Binding of anti-cyclopeptide antibodies to oligomeric amyloid β (Aβ) as determined by ELISA.

<p>Threefold serial dilutions (starting from 1/150) of pooled antisera obtained were tested. Antisera were raised by immunization with oligomeric (black ▴) or fibrillar (black ▪) Aβ(1–42) and TTd-conjugates of cyclo[Aβ(22–28)-YNGK′] (red ▴) or linear K′-Aβ(22–28)-YNG (red ▪). Monoclonal antibody 6E10 (green •) served as control. Antibody binding to fibrillar Aβ(1–42) gave comparable results.</p