The relative utilization of alternative processing pathways for APP can be regulated by the activation state of certain protein phosphorylation signal transduction pathways. For example, activation of protein kinase C (PKC), or inactivation of protein phosphatases 1 and 2A leads to a relative increase in utilization of the non-amyloidogenic alpha secretase cleavage pathway for APP processing at the expense of other pathways. Current data suggest that major candidate phosphorylation-state sensitive targets relevant to the molecular basis of PKC-activated processing (or """"""""regulated cleavage"""""""") of APP include secretase enzymes and/or other components of the APP trafficking/processing apparatus. Further efforts to distinguish among these possibilities will be investigated during the next award period and are summarized here. One approach will be the in vitro reconstitution in manipulatable systems of certain events relevant to the regulated cleavage of APP. (Specific aim I). A porated cell system has been developed that provides for the selective study of APP processing events at the plasma membrane in the absence or presence of cytosolic components. A second system that has been developed involves the study of the regulation of formation of nascent constitutive secretory vesicles containing APP. Another approach involves the study of metabolism of APP in Saccharomyces cerevisiae (Specific Aim II). We have demonstrated that alpha-secretase like events occur in Saccharomyces and we now are poised to study how PKC regulates normal and sec mutant constitutive pathways in Saccharomyces. These approaches in vitro reconstituted systems and Saccharomyces will be complemented by sub-cellular fractionation studies in control and drug-treated intact mammalian cells (Specific AIm III). Finally, in addition to the forgoing studies which focus primarily on alpha-secretase cleavage of APP, we also propose to study the role of the multivesicular body in the gamma-secretase cleavage of APP which gives rise to the carboxy-terminus of Abeta and p3 (Specific Aim IV). A clear elucidation of the molecular and cellular basis for these features of APP metabolism could form the basis of a rational therapeutic strategy for diminishing the cerebral amyloid deposition which accompanies Alzheimer's Disease.
