CHEMISTRY AND BIOLOGY OF ALZHEIMER'S AMYLOID PROTEINS

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DESCRIPTION: (adapted from Applicant's Abstract) The long-term goal of this study is to establish the role that amyloid plays in the pathology of Alzheimer's Disease (AD). Despite the advances made by numerous laboratories, the exact chemical nature of the amyloid minor protein components still remains to be elucidated. It is hypothesized that these ancillary beta amyloid (AB) proteins are of great significance in amyloid deposition and in the pathogenesis of AD. Therefore, this study specifically aims to purify and determine the primary structures and post-translational modifications of the major and minor amyloid components from neuritic plaques vascular walls and diffuse deposits. Ultra structural immunochemistry will be utilized to ascertain the location of the minor amyloid components in these deposits. The biological effects of neuritic plaque, vascular amyloid, diffuse amyloid and their separated components will be assessed on neuronal and glial cells in culture. To achieve these goals, amyloid will be purified from AD brain homogenates as well as from blood vessels by recently developed protocols which include filtration, isocratic sucrose separations, ultracentrifugation, collagenase hydrolysis, detergent lysis and discontinuous sucrose density gradients. Solubilization of the amyloid proteins by denaturing, chaotropic and dispersing agents will be followed by their purification using multiple steps of high performance liquid chromatography (HPLC). Antisera will be generated against the different separated protein fractions and used in light and electron microscopic immunochemical (colloidal gold) studies to determine their location in situ and in purified amyloid.The relevant proteins and derived enzymic and chemical peptides will be further fractionated by HPLC and characterized by automatic amino acid analysis and microsequencing as well as by mass spectrometry. Determination of post- translational modifications will be carried out by gas chromatography- mass spectrometry and by two-dimensional high voltage electrophoresis. Elucidation of the chemical nature and of the biological actions of amyloid proteins will contribute to the understanding of the pathogenesis and pathophysiology of AD. This knowledge will also assist in the development of molecular models for AD and will aid in the implementation of therapeutic strategies.