Beta-amyloid protein (Abeta), the major constituent of the fibrils composing senile plaques and amyloid deposits in cerebral blood vessels in AD and other beta-amyloid related disorders, is an aberrant degradation product of a larger precursor APP. The deposition of Abeta in cerebral vessel wall may involve a complex interaction of different cell types and various proteins. The comprehension of the mechanisms by which Abeta is processed from its precursor and the determination of the extent to which early and mature plaque formation contribute to neuronal cell damage and disease progression remain central, important questions in the understanding of AD. The cellular origin of the APP is controversial and based on biochemical, immunohistological and DNA/RNA studies, two different sources for APP have been proposed: neuronal and vascular. In support to the vascular theory we have observed that, i) in Dutch patients with familial cerebral amyloid angiopathy or hereditary cerebral hemorrhage (HCHWA-D), amyloid deposits are largely restricted to leptomeningeal and cortical vessel walls, and neuritic plaques as well as neurofibrillary tangles are absent; ii) in HCHWA-D and in AD patients, Abeta and APP coexist in the same vessel, suggesting that the processing of APP into insoluble fibrils may take place """"""""in situ""""""""; iii) platelets contain intact APP in the alpha-granules, which can be released upon thrombin activation, indicating the potential importance of these cells as a source for APP. Because of these findings, we propose: I) to study the processing of APP in vessel wall by using purified brain vessels from AD and controls as well as brain endothelial and smooth muscle cells in culture and platelets. Synthetic peptides homologous to APP sequences comprising the Abeta N- and C-terminus will be used as substrates, as well as soluble APP (from platelets and CSF), full length APP (purified from Baculovirus infected Sf9 cells expressing recombinant APP751) and fragments containing the entire amyloid region (purified from Baculovirus infected cells; see Core). II) to purify and biochemically characterize the enzyme(s) from vascular wall and/or cell component(s) involved in the normal and abnormal APP processing. III) once the enzyme(s) involved in the processing are characterized, the development of specific protease inhibitors will be attempted.
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