The broad, long-term objective of this proposal is to contribute to our understanding of the molecular basis of Alzheimer disease (AD). This objective will be advanced by characterizing APP in human platelets, by determining how APP is metabolized, and by relating these findings to the pathogenesis of AD. The four specific aims of this proposal are: 1) To study the species (isoforms and/or proteolytic fragments) of APP present in human platelets; 2) To investigate how APP is processed in human platelets, including determining which signal transduction mechanisms initiate processing, and which enzymes catalyze APP cleavage; 3) To establish human platelets as an in vitro model for APP processing with the intention of using it for testing agents that might modulate processing; 4) To study whether processing of APP in human platelets can generate APP metabolites that might be involved in AD-associated amyloidosis. The health-relatedness of this project is its aim of identifying the causes of AD and effective therapeutics for the AD- associated amyloidosis. The experimental design for these studies is to identify APP isoforms and metabolites in human platelets using antibodies with specificities for different regions of the APP molecule as well as antibodies against different isoforms of APP. Metabolism of APP will be studied in platelets with and without stimulation by physiological and pharmacological agents that induce degranulation. The subcellular distribution of APP and APP-metabolites will be localized using subcellular fractionation techniques. Phosphorylation and dephosphorylation as processes which might regulate APP metabolism will be studied. The enzyme(s) which are responsible for the proteolytic processing of APP will be purified and characterized. Metabolites of APP that possibly can be involved in Beta/A4-amyloidogenesis and pathogenesis of AD will be identified by purification and limited amino- terminal sequencing of APP metabolites from stimulated and non- stimulated platelets.
