Design, Characterization, and Use of a Novel Amyloid ß-Protein Control for Assembly, Neurotoxicity, and Gene Expression Studies.

ABSTRACT

A key pathogenic agent in Alzheimer's disease (AD) is the amyloid ß-protein (Aß), which self-assembles into a variety of neurotoxic structures. Establishing structure-activity relationships for these assemblies, which is critical for proper therapeutic target identification and design, requires aggregation and neurotoxicity experiments that are properly controlled with respect to the Aß peptide itself. "Reverse" Aß or non-Aß peptides suffer from the fact that their biophysical properties are too similar or dissimilar, respectively, to those of native Aß for them to be appropriate controls. For this reason, we used simple protein design principles to create scrambled Aß peptides predicted to behave distinctly from native Aß. We showed that our prediction was true by monitoring secondary structure dynamics with thioflavin T fluorescence and circular dichroism spectroscopy, determining oligomer size distributions, and assaying neurotoxic activity. We then demonstrated the utility of the scrambled Aß peptides by using them to control experiments examining the effects of Aß monomers, dimers, higher-order oligomers, and fibrils on gene expression in primary rat hippocampal neurons. Significant changes in gene expression were observed for all peptide assemblies, but fibrils induced the largest changes. Weighted gene co-expression network analysis revealed two predominant gene modules related to Aß treatment. Many genes within these modules were associated with inflammatory signaling pathways.