Alzheimer's disease (AD) is a disorder characterized by abnormal protein phosphorylation and altered protein catabolism. Research focussing on the regulation of phosphorylation of AD-related proteins may help elucidate the exact defect(s) responsible for the disease. In this application, we propose to study the regulation of phosphorylation of substrate phosphoproteins relevant to the pathobiology of AD. These proteins are (i) the Alzheimer amyloid precursor protein (APP); (ii) its Drosophila homologue APP-like, or APPL; and (iii) APP-promoter binding homeoproteins, which may, in turn, regulate APP expression. Complementary studies will be carried out both in vitro using purified reaction components and in a variety of intact cell systems. The intact cell systems will include isolated intact nerve terminals, brain slices, and cells from continuous or primary neuronal culture (rodent-derived for APP, fly-derived for APPL). DNA transfer techniques will be utilized at times to modify the intact systems in a discrete fashion. Cells from continuous culture will be transfected with native or mutant forms of APP or APPL. These mutations will be designed primarily for the purpose of identifying the phosphorylated residues in the cytoplasmic domains of these proteins. Primary neuronal cultures will be derived from the brains of normal rats and mice, and from the brains of transgenic mice, prepared by the Core. These mice will overexpress APP isoforms or mutant isoforms, or AD/APP- related protein kinases. First messengers (neurotransmitters, neuropeptides, growth factors) will be screened as candidate activators of phosphorylation of APP, APPL and homeoproteins. Membrane-permeable second messengers and agents acting directly on protein kinases or protein phosphatases will also be surveyed. Data from these enzymological experiments will complement the design and interpretation of cell biological (Project 2), anatomical (Project 3) and molecular biological (Project 4) studies proposed by other investigators elsewhere in the Program. Another major focus of this Project is to define and characterize the changes in protein phosphorylation which accompany Alzheimer-type pathology in the human brain. A comprehensive and systematic survey of phosphorylation systems and their activities will be performed in the normal young, normal aged and Alzheimer brain. Enzymological and immunochemical approaches will be used in Project 1, complementing anatomical approaches to be undertaken by Project 3. In summary, Alzheimer's disease is characterized by intracellular accumulation of cytoskeletal phosphoproteins and the extracellular deposition of a portion of an integral membrane phosphoprotein. The precise role of protein phosphorylation in the pathobiology of Alzheimer's disease is not known but can be sought at the molecular, biochemical and cell biological levels. Data from these experiments should offer insights which are likely to be of clinical and therapeutic importance.