Using Chirality to Understand and Control Amyloid Beta Neuronal Uptake and Toxicity

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ABSTRACT Amyloid β (Aβ) is a believed key toxic agent of Alzheimer’s Disease (AD). To develop AD therapeutics, an improved understanding of the mechanisms of Aβ toxicity is urgently needed. In the brain, Aβ is found mostly in extracellular deposits that may be taken up by neurons. The purpose of this proposal is to test the central hypothesis that neuronal Aβ uptake and toxicity are linked. Aβ forms diverse aggregates with varied neurotoxic profiles. Little is known about how structure and aggrega- tion state affect neuronal uptake and toxicity of Aβ. This makes it very difficult to devise strategies to block these pathogenic processes. Efforts to determine how conformation and aggregation state of Aβ affects its’ neuronal uptake and toxicity were hampered thus far by the lack of (a) methods to produce stable samples for structural analysis and (b) accurate tools to quantify neuronal uptake of different Aβ aggregates. Recent chirality-based approaches of the Raskatov lab have produced a set of stabilized oligomeric and fibrillary Aβ forms that will be used here as tools, with the goal to close this important knowledge gap. Proposed research is cross-disciplinary and collaborative: it includes structural collaborations with Dr. Eisenberg and Dr. Tycko; Dr. Glabe will consult on neurobiology experiments done in the Raskatov lab. The purpose of Aim 1 is to complete the structural elucidation of racemic Aβ fibrils by ssNMR, to then use those structural insights to devise smaller, more drug-like, oligomer-to-fibril converters, and to test the working hypothesis that oligomer-to-fibril conversion reduces Aβ uptake into neurons, thus suppressing its toxicity. This will be accomplished using C14-based radioquantitation tools in combination with various cell culture assays to measure both rapid and slow toxic actions of Aβ against neurons. Aim 2 will test the working hypothesis that the differences in toxicity between Aβ42-E22e and Aβ42-S26s are due to differences in their neuronal uptake, and will also test the alternative hypothesis that the peptides traffic to different sub-cellular sites, and that the differences in peptide toxicity are due to that. CryoEM structures of Aβ42-E22e and Aβ42-S26s stabilized oligomers will be sought, to identify the structural motifs responsible for their toxicity differences. Aim 3 will test the working hypothesis that the highly aggregation-prone, N-terminally truncated Aβ-related peptide p3 promotes oligomer-to-fibril conversion in Aβ, thus reducing Aβ uptake efficiency and making it less neurotoxic. Successful completion will yield a quantitative link between neuronal uptake and toxicity of different Aβ forms. It may yield the world’s first Aβ oligomer structures, as well as a structure of non-toxic Aβ fibrils. It may yield smaller, D-peptidic Aβ oligomer-to-fibril converters to be translated to novel AD therapeutics in the future, and it will also reveal how Aβ toxicity is suppressed by p3 addition. Finally, the proposed studies may uncover general structural insights on how protein aggregation affects neuronal uptake and toxicity.