Secretory cleavage of the amyloid precursor protein (APP) of Alzheimer's disease (AD) is a regulated process that increases the release of soluble APP derivatives (sAPP), molecules with neurotrophic and neuroprotective properties, and inhibits formation of neurotoxic, amyloid- forming Abeta proteins. Maintaining sAPP release, and decreasing Abeta formation, may be key factors in preventing neuronal degeneration and amyloid deposition in AD; and one of the principal goals of this application is the elucidation of intracellular signaling pathways that control these processes. Previous results indicate that sAPP release elicited by muscarinic receptor activation is reduced by inhibition of protein kinase C (PKC) and tyrosine kinases, and is correlated with tyrosine phosphorylation of several proteins including paxillin and focal adhesion kinase (FAK). Activation of the epidermal growth factor (EGF) receptor excerts similar effects on sAPP release and tyrosine phosphorylation; both effects are reduced kinase inhibitor.
Aim 1 of this proposal is designed to identify specific non-receptor tyrosine kinases that regulate APP processing (sAPP release and Abeta formation) and tyrosine phosphorylation in response to muscarinic and EGF receptor activation, using dominant negative mutant Src, FAK and Pyk2 proteins and in vitro kinase assays. Paxillin and FAK are components of focal adhesions,- sites at which cell surface integrins bind to extracellular matrix (ECM) proteins,- and are tyrosine phosphorylated during cell adhesion, as well as following receptor activation. Evidence presented in this proposal indicates that cell adhesion to the ECM protein fibronectin stimulates sAPP release and tyrosine phosphorylation, and is accompanied by an apparent interaction between APP and the integrin receptor. Experiments proposed in aim 2 will examine the role played by integrin activation and tyrosine kinases in the stimulation of APPs release by cell adhesion to fibronectin, and will test the hypothesis that a physical association of APP with the integrin receptor occurs in cells adherent to fibronectin. Immunoprecipitation and immunofluorescence approaches, and analyses with PKC and tyrosine kinase inhibitors, and dominant negative tyrosine kinase mutants, will be used to examine these interactions. Another goal of aim 2 will be to test the hypothesis that soluble Abeta peptides inhibit muscarinic receptor-coupled tyrosine phosphorylation by interfering with integrin activation. Finally, in aim 3, regulation of the putative alpha-secretase known as Tumor Necrosis Factor-alpha Converting Enzyme (TACE) by muscarinic receptor-mediated phosphorylation will be examined using a co-transfection strategy. The proposed experiments will establish the fundamental role of tyrosine kinases in the control of APP processing.
Showing the most recent 10 out of 11 publications
