Helical sulfonyl-γ-AApeptides modulating Aß oligomerization and cytotoxicity by recognizing Aß helix.

In contrast to prevalent strategies which make use of ß-sheet mimetics to block Aß fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aß13-26 and stabilized Aß conformation to avoid forming the neurotoxic Aß oligomeric ß-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aß oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aß42 and induce α-helix conformation in Aß. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aß aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aß-mediated cytotoxicity even in the presence of pre-formed Aß aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aß42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aß42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aß into the off-pathway helical secondary structure, thereby preventing the formation of Aß oligomerization, fibrillation and rescuing Aß induced cell cytotoxicity.

Crystal Violet Selectively Detects Aß Oligomers but Not Fibrils In Vitro and in Alzheimer's Disease Brain Tissue.

Deposition of extracellular Amyloid Beta (Aß) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aß and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aß42 oligomers (AßOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.

Self-Assembly of Amyloid Fibrils into 3D Gel Clusters versus 2D Sheets.

The deposition of dense fibril plaques represents the pathological hallmark for a multitude of human disorders, including many neurodegenerative diseases. Fibril plaques are predominately composed of amyloid fibrils, characterized by their underlying cross beta-sheet architecture. Research into the mechanisms of amyloid formation has mostly focused on characterizing and modeling the growth of individual fibrils and associated oligomers from their monomeric precursors. Much less is known about the mechanisms causing individual fibrils to assemble into ordered fibrillar suprastructures. Elucidating the mechanisms regulating this "secondary" self-assembly into distinct suprastructures is important for understanding how individual protein fibrils form the prominent macroscopic plaques observed in disease. Whether and how amyloid fibrils assemble into either 2D or 3D supramolecular structures also relates to ongoing efforts on using amyloid fibrils as substrates or scaffolds for self-assembling functional biomaterials. Here, we investigated the conditions under which preformed amyloid fibrils of a lysozyme assemble into larger superstructures as a function of charge screening or pH. Fibrils either assembled into three-dimensional gel clusters or two-dimensional fibril sheets. The latter displayed optical birefringence, diagnostic of amyloid plaques. We presume that pH and salt modulate fibril charge repulsion, which allows anisotropic fibril-fibril attraction to emerge and drive the transition from 3D to 2D fibril self-assembly.

Origin, toxicity and characteristics of two amyloid oligomer polymorphs.

There is compelling evidence that small oligomeric aggregates, emerging during the assembly of amyloid fibrils and plaques, are important molecular pathogens in many amyloid diseases. While significant progress has been made in revealing the mechanisms underlying fibril growth, understanding how amyloid oligomers fit into the fibril assembly process, and how they contribute to the pathogenesis of amyloid diseases, has remained elusive. Commonly, amyloid oligomers are considered to be metastable, early-stage precursors to fibril formation that are either on- or off-pathway from fibril growth. In addition, amyloid oligomers have been reported to colocalize with late-stage fibrils and plaques. Whether these early and late-stage oligomer species are identical or distinct, and whether both are relevant to pathogenesis remains unclear. Here we report on the formation of two distinct oligomer species of lysozyme, formed either during the early or late-stages of in vitro fibril growth. We further observe that the pH change from in vitro growth conditions to cell media used for toxicity studies induced distinct mesoscopic precipitates, two of which resemble either diffuse or neuritic plaques seen in Alzheimer's histology. Our biophysical characterization indicates that both oligomer species share morphological and tinctorial features considered characteristic for amyloid oligomers. At the same time, their sizes, morphologies, their immunostaining, detailed tinctorial profiles and, most prominently, their biological activity are clearly distinct from each other. Probing the conditions promoting the formation of these two distinct oligomer species suggests distinct roles of charge interactions, hydrophobicity and monomer flexibility in directing oligomer assembly.