Structural and Biological Characterization of Diverse Oligomers Derived from Abeta

University Of California-Irvine, Irvine CA

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Abstract

Project Summary/Abstract: Understanding the aggregation of the ?-amyloid peptide (A?) to form toxic oligomers is fundamental to understanding the molecular basis of Alzheimer?s disease (AD). Despite decades of research, the structures of A? oligomers remain a mystery, constituting a significant gap in understanding AD. This proposal seeks to address this knowledge gap through the structural, biophysical, and biological profiling of a diverse group of A? oligomer models and correlation of these models with biogenic A? oligomers. My laboratory has developed an approach for create structurally defined A? oligomer models composed of peptide fragments from A? constrained into a ?-hairpin. The X-ray crystallographic structures of these A? ?- hairpin peptides reveal the structures of oligomers that the peptides can form and key intermolecular contacts that the peptides make in the oligomeric state. These contacts reveal sites that can be crosslinked to create covalently stabilized A? oligomer models that mimic the crystallographic oligomers. Studying the crosslinked oligomers then allows detailed correlation between oligomer structure and biophysical and biological properties. We will characterize how our A? oligomer models interact with and affect neurons, microglia, and astrocytes, to provide detailed insights into how our A? oligomer models impact different brain cell types and thus help shed light on the relationship between A? oligomer structure and cellular events that occur in AD. We will use fluorescence microscopy and fluorescence-assisted cell sorting (FACS) to visualize and quantify the interactions and uptake of our A? oligomer models with neurons, microglia, and astrocytes. We will evaluate downstream biochemical and cellular effects elicited by A? oligomer models in neurons, microglia, and astrocytes and evaluate how treatment affects apoptosis, necrosis, calcium homeostasis, endoplasmic reticulum stress, oxidative stress, neurite length, tau phosphorylation and aggregation, and proinflammatory responses in microglia and astrocytes. To elucidate the relationship between the structures of our A? oligomer models and biogenic A? oligomers, we will generate polyclonal antibodies against each A? oligomer model and then examine the immunoreactivity of these antibodies with brain protein extract and brain slices from 5XFAD mice. We will discover new A? oligomer models, by creating new A? ?-hairpin peptides that contain more of the A? peptide sequence and alternate ?-strand alignments. We will create new crosslinked A? oligomer models by identifying key contacts in existing and newly discovered A? oligomer models and then engineering in disulfide bonds to stabilize the oligomers. To characterize the structures and oligomerization properties of the new A? oligomer models that we generate, we will use X-ray crystallography and a variety of other biophysical experiments, including CD spectroscopy, SDS-PAGE, size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), NMR spectroscopy, and Förster resonance energy transfer (FRET) studies. The biological and immunological properties of these new A? oligomer models will be studied as described above.