Molecular Organization of Alzheimer abnormal Fibers

        Abnormal filamentous structures accumulate both inside neurons (in neurofibrillary tangles and the neurites of senile plaques) and extracellularly (in senile plaque cores and blood vessel walls) in Alzheimer’s disease. The individual filaments are on the order of 80 Angstroms thick (or three ten millionths of an inch), and their presence signals disruption and degeneration of neurons. To understand how and why these assemblies form requires an understanding of their structural organization at the molecular level. The major consti- tuent of some of these filaments is a protein called amyloid. Because progressive amyloid deposition seems to play a central role in the pathogenesis of AD, we plan to study the detailed molecular structure of the macromolecular assemblies formed by this protein. Determination of the organization of the structural units that comprise the abnormal  filaments will illuminate our understanding of how the abnormal filaments might be related to or derive from normally occurring cellular proteins and structural components in the neuron.                    The mechanism by which proteins are compelled by the sequence if their amino acids to fold into three-dimensional structures and to assemble into larger aggregates is one of the fundamental problems in medical/biochemical research. It assembled amyloid fibrils the predominant structural motif is the so-called cross-ß fibril. In this structure, the protein chains are oriented at right angles to the fibril direction. The chains are stabilized in the fibril direction by bonds between hydrogen atoms to form extended ß-sheets, and the sheets in turn are stabilized to one another by hydrophobic and electrostatic interactions. Such ß-sheets also occur frequently in globular proteins or enzymes. The amyloid protein provides a superb model system for determining what are the most important factors that direct assembly of a polypeptide into a cross-ß structure rather than into an extended-ß, a-helix, random coil. In the case of Alzheimer amyloid, based on the results of our fundamental research into the factors that stabilize these proteins into insoluble fibers, it may become possible eventually to devise methods of preventing their formation or  disrupting these assemblies in the afflicted individual.          X-ray diffraction is a technique uniquely suited for characterizing the three- dimensional structure of ordered assemblies. We will use this method to examine the ability of synthetic polypeptides that have amino acid sequences resembling the Alzheimer amyloid protein to form fibrils. Modifications of the synthetic polypeptides, for example by altering the sequence of their amino acids, will enable us to identify what molecular features are crucial in promoting or preventing the formation and accumulation of fibrils. To complement the X-ray findings, we will use electron microscopy to examine fibril morphology. We will also use Fourier transform infra-red spectroscopy to study the  conformation of the synthetic polypeptides in solution. Such information will help to determine what portion of the polypeptide is incorporated into the ß-pleated sheet, what type of folding is assumed by those portions of the chain not involved in the ß-sheet, and what conditions are required for fibril assembly (e.g., salt concentration, specific metals, and amount of acidity if alkalinity). Computer graphics will be used to build models of  the polypeptides and to indicate possible interactions that could generate fibrils from them. Finally, results on the reconstituted fibrils will be compared with findings on native fibrils isolated from Alzheimer disease brain tissue.                        In summary, our findings will address the mechanism whereby the amyloid protein assembles into insoluble fibrous bundles; the requisite sequence specificity for forming their particular protein folding; and the molecular interactions that stabilize the amyloid peptides as virtually insoluble fibrils. A detailed understanding of the three-dimensional organization in these abnormal fibers will complement other information currently being obtained from analytical protein chemistry and immunocytochemistry, and will provide valuable insights into the pathogenesis of neurofibrillary tangles and neuritic plaques in normal human aging, in Alzheimer’s disease, and in related neuronal degenerative disorders. Further, our findings may have broader implications  regarding the formation of insoluble fibrous assemblies from other aging-related amyloid proteins, for example that occur in the amyloidoses.