Immobilization of Homogeneous Monomeric, Oligomeric and Fibrillar Aβ Species for Reliable SPR Measurements

There is strong evidence that the amyloid-beta peptide (Aß) plays a central role in the pathogenesis of Alzheimer’s disease (AD). In this context, a detailed quantitative description of the interactions with different Aß species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aß in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aß conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aß allows reliable SPR measurements and quality control of the immobilized Aß aggregate species at any step throughout the experiment

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ1–42 species, reduced Aβ1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein

Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized D-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified D-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD

A D-enantiomeric peptide interferes with hetero-association of amyloid-β oligomers and prion protein

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of amyloid-β (Aβ) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression. Aβ oligomers bind with high affinity to membrane-associated prion protein (PrP), leading to toxic signaling across the cell membrane, which makes the Aβ–PrP interaction an attractive therapeutic target. Here, probing this interaction in more detail, we found that both full-length, soluble human (hu) PrP(23–230) and huPrP(23–144), lacking the globular C-terminal domain, bind to Aβ oligomers to form large complexes above the megadalton size range. Following purification by sucrose density–gradient ultracentrifugation, the Aβ and huPrP contents in these hetero-assemblies were quantified by reversed-phase HPLC. The Aβ:PrP molar ratio in these assemblies exhibited some limited variation depending on the molar ratio of the initial mixture. Specifically, a molar ratio of about four Aβ to one huPrP in the presence of an excess of huPrP(23–230) or huPrP(23–144) suggested that four Aβ units are required to form one huPrP-binding site. Of note, an Aβ-binding all-D-enantiomeric peptide, RD2D3, competed with huPrP for Aβ oligomers and interfered with Aβ–PrP hetero-assembly in a concentration-dependent manner. Our results highlight the importance of multivalent epitopes on Aβ oligomers for Aβ–PrP interactions and have yielded an all-D-peptide-based, therapeutically promising agent that competes with PrP for these interactions

On the Examination of the Strong Discontinuity Analysis for a Kinking Discontinuous Surface

The purpose of this paper is to examine the accuracy of the analysis of the strong discontinuity with a kinking discontinuous surface. We first examine the path independent J-integral and the E-integral formula for a damage model with a kinking cohesive region. As a result, we find that the strong discontinuity analysis based on the E-integral has a high accuracy in view of the energy release rate even when the discontinuous surface kinks

A mirror-phage display selected D-peptide and its derivative eliminate Abeta oligomers in vitro and decelerate cognitive and motoric impairments of transgenic AD mice in vivo

Several lines of research provide strong evidence for a central role of amyloid-beta (Abeta) oligomers in the pathogenesis of Alzheimer's disease. Investigations on Abeta oligomer interference, however, are impeded by the lack of a quantitative assay to measure substance-induced effects on Abeta oligomers. We have developed a comprehensive, fast and reliable in vitro assay to quantify the removal of Abeta oligomers by any potential drug, for example D3 and its dimeric form, denominated D3D3. This multivalent D3 molecule was expected to have enhanced efficacy due to increased avidity. Therefore we wanted to investigate if there is a correlation between the removal of Abeta oligomers by D3 and D3D3 and positive effects on cognitive and motoric performance in transgenic AD animal models

Interaction of scFv-IC16 with different immobilized Aβ(1–42) assembly states.

<p>SPR sensorgrams were recorded separately with single-cycle kinetics. Experimentally obtained, double-referenced binding data (black traces) were superimposed with the corresponding fit (red traces). Monomer data was fit to a 1∶1 Langmuir binding model, and oligomer and fibril data were fit to a heterogeneous ligand binding model. ΔRU: delta of the response units. t/s: time in seconds.</p

Kinetic rates obtained for scFv-IC16 binding to different immobilized Aβ(1–42) assembly states.

<p>Association rate constants (<i>k</i>_a) were plotted against dissociation rate constants (<i>k</i>_d). The dissociation constant (<i>K</i>_D) can be extracted from the diagonal lines. Circles, squares and triangles correspond to data from interactions with monomers, oligomers and fibrils, respectively, whereas filled symbols represent data for the second binding site. All data was determined with the heterogeneous fitting model. The grey circle represents monomer data obtained with a 1∶1 binding model.</p

Overview of kinetic rates for scFv-IC16 binding to different Aβ(1–42) assembly states obtained within the single-cycle kinetic SPR experiments.

<p>The hash (^#) denotes that kinetic rates were determined with a heterogeneous binding model. Standard deviation with number of experiments is given in brackets.</p>#<p>fit to a heterogeneous binding model. Units are: [a] Ms⁻¹, [b] s⁻¹, [c] M, and [d] RU.</p