The overall goals of this proposal are to understand the structure of the Amyloid Ab peptide and the dynamics of its assembly into amyloid fibrils that are characteristic of Alzheimer's disease (AD). The mechanisms of action of other macromolecules that may promote or inhibit assembly and deposition in amyloid plaque will be determined. Studies on the effects of mutations associated with familial forms of AD on the production of Ab implicated amyloid as a causative agent in the etiology of AD. Results from studies on Ab toxicity in cell culture systems suggest that the aggregation and self assembly properties of Ab may also play a critical role in the biological activity of Ab. The first specific aims is to determine the structure of Ab in solution and when it is assembled into amyloid fibrils. Distances between different points along the Ab sequence will be measured in the oligomeric and fibrillar state by resonance energy transfer and chemical crosslinking. We have developed a set of fluorescent Ab analogs and amino acid substitution analogs that display the same assembly properties as wild type Ab for use in these studies. The location of solvent exposed amino acid residues and position residing in polar and non-polar environments will be probed by fluorescence spectroscopy, quenching analysis and reactivity with polar thiol-reactive agents analogous to the strategy to map the polar surface of acetylcholine receptor channel. These studies should provide information about the spatial relationship between different points along the peptide backbone and will be critical to validate or rule out structures obtained by molecular modeling of NMR analysis of smaller fragments of Ab. This information may ultimately be useful in conceptualizing strategies and therapeutic agents to interfere with Ab aggregation and understanding the relationship between Ab structure and cytotoxic activity. In the second aim, we will characterize oligomeric aggregates that may represent intermediates in the fibril assembly reaction and determine which steps of the assembly process are rate limiting. We will examine whether the fibril elongates equally at both ends. We will investigate whether the dynamics of assembly are altered under different conditions that may be physiologically relevant to amyloid accumulation. In the third specific aims, we will characterize the mechanism of action of a number of potential inhibitors and effectors of Ab assembly that have been proposed to play a role in the deposition of amyloid in AD. We hope to identify classes of molecules that specifically inhibit or promote several different, critical steps in Ab fibril assembly such as nucleation of fiber assembly. Knowing the specific effects of the various molecules and the concentration dependence will provide a means of evaluating the potential relevance in amyloid deposition; strengthening the relevance of molecules that are effective under conditions that are believed to prevail in vivo and weakening the relevance of molecules that would not be expected to function under physiological conditions.
| Mamikonyan, Grigor; Necula, Mihaela; Mkrtichyan, Mikayel et al. (2007) Anti-A beta 1-11 antibody binds to different beta-amyloid species, inhibits fibril formation, and disaggregates preformed fibrils but not the most toxic oligomers. J Biol Chem 282:22376-86 |
