The hallmark morphological abnormalities of Alzheimer's disease (AD) are deposits of amyloid fibers, which constitute neuritic plaques and cerebrovascular amyloid, and paired helical filaments (PHF), which constitute neurofibrillary tangles. The formation and accumulation of these fibrous assemblies profoundly affect memory, language and behavior. Our research is focused on the major protein of AD amyloid, called beta/A4, and on the microtubule-associated protein tau, which is a major cytoskeletal protein and an integral component of PHF. Using a correlation of results from X-ray diffraction, electron microscopy and Fourier-transform infrared spectroscopy, we plan to provide a detailed description of the molecular and macromolecular structures of the fibrous assemblies of AD. In addition, we will determine the forces that promote their formation, the factors that underlie their stability, and the specific residues that promote their folding. To accomplish our objective, this proposal focuses on the following three Specific Aims: (1) To test specific hypotheses regarding the self-assembly and stability of synthetic beta/A4 homologues, and their interactions with tissue components that are thought to promote their formation. The hypotheses are: (i) electrostatic interactions involving the formation of a salt-bridge within the core region of beta/A4 are crucial in stabilizing the beta-pleated sheets of the fibrils; (ii) a single amino acid residue change in the sequence of beta/A4 accounts for the extremely high level of aggregation of amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of the Dutch type; (iii) alpha- anti-chymotrypsin (ACT) binds specifically to and alters the structure of beta/A4, thus affecting its proteolytic processing; and (iv) sulfated proteoglycans of brain extracellular matrix promote the polymerization and/or aggregation of AD amyloid by electrostatic interactions involving the sulfate groups. (2) To investigate the assembly of PHF from modified forms of microtubule-associated tau protein. A microdialysis technique originally developed for crystallizing membraneproteins will be used to manipulate and control the gradual formation of PHF-like filaments. Suitable samples will be subjected to X-ray diffraction, which is expected to provide new details of PHF organization that has so far resisted such analysis. (3) To develop molecular models of these fibrillar assemblies based on constraints imposed by the biophysical and ultrastructural results. We will continue to develop analytical tools that will be used to interpret the fiber diffraction patterns and to model details of fibril organization at the molecular level. In achieving our objective, we hope to develop sufficient understanding for the eventual rational development of diagnostic/therapeutic strategies for AD, for related neurodegenerative diseases, and for amyloidoses in general.
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