EPR Studies of Aß42 Oligomers Indicate a Parallel In-Register ß-Sheet Structure.

Aß aggregation leads to the formation of both insoluble amyloid fibrils and soluble oligomers. Understanding the structures of Aß oligomers is important for delineating the mechanism of Aß aggregation and developing effective therapeutics. Here, we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study Aß42 oligomers prepared by using the protocol of Aß-derived diffusible ligands. We obtained the EPR spectra of 37 Aß42 oligomer samples, each spin-labeled at a unique residue position of the Aß42 sequence. Analysis of the disordered EPR components shows that the N-terminal region has a lower local structural stability. Spin label mobility analysis reveals three structured segments at residues 9-11, 15-22, and 30-40. Intermolecular spin-spin interactions indicate a parallel in-register ß-sheet structure, with residues 34-38 forming the structural core. Residues 16-21 also adopt the parallel in-register ß-structure, albeit with weaker intermolecular packing. Our results suggest that there is a structural class of Aß oligomers that adopt fibril-like conformations.

The supersaturation perspective on the amyloid hypothesis.

Development of therapeutic interventions for Alzheimer's over the past three decades has been guided by the amyloid hypothesis, which puts Aß deposition as the initiating event of a pathogenic cascade leading to dementia. In the current form, the amyloid hypothesis lacks a comprehensive framework that considers the complex nature of Aß aggregation. The explanation of how Aß deposition leads to downstream pathology, and how reducing Aß plaque load via anti-amyloid therapy can lead to improvement in cognition remains insufficient. In this perspective we integrate the concept of Aß supersaturation into the amyloid hypothesis, laying out a framework for the mechanistic understanding and therapeutic intervention of Alzheimer's disease. We discuss the important distinction between in vitro and in vivo patterns of Aß aggregation, the impact of different aggregation stages on therapeutic strategies, and how future investigations could integrate this concept in order to produce a more thorough understanding and better treatment for Alzheimer's and other amyloid-related disorders.

A protein aggregation platform that distinguishes oligomers from amyloid fibrils.

Deposition of aggregated proteins is a pathological feature in many neurodegenerative disorders such as Alzheimer's and Parkinson's. In addition to insoluble amyloid fibrils, protein aggregation leads to the formation of soluble oligomers, which are more toxic and pathogenic than fibrils. However, it is challenging to screen for inhibitors targeting oligomers due to the overlapping processes of oligomerization and fibrillization. Here we report a protein aggregation platform that uses intact and split TEM-1 ß-lactamase proteins as reporters of protein aggregation. The intact ß-lactamase fused with an amyloid protein can report the overall protein aggregation, which leads to loss of lactamase activity. On the other hand, reconstitution of active ß-lactamase from the split lactamase construct requires the formation of amyloid oligomers, making the split lactamase system sensitive to oligomerization. Using Aß, a protein that forms amyloid plaques in Alzheimer's disease, we show that the growth curves of bacterial cells expressing either intact or split lactamase-Aß fusion proteins can report changes in the Aß aggregation. The cell lysate lactamase activity assays show that the oligomer fraction accounts for 20% of total activity for the split lactamase-Aß construct, but only 3% of total activity for the intact lactamase-Aß construct, confirming the sensitivity of the split lactamase to oligomerization. The combination of the intact and split lactamase constructs allows the distinction of aggregation modulators targeting oligomerization from those targeting overall aggregation. These low-cost bacterial cell-based and biochemical assays are suitable for high-throughput screening of aggregation inhibitors targeting oligomers of various amyloid proteins.