Mechanisms underlying the age-related decline in cognitive performance and increased susceptibility to neurodegenerative diseases are not known, though dysregulation of Ca2+ homeostasis and enhanced oxidative stress appear to be primary contributors. We have found that a major Ca2+ regulator, the plasma membrane Ca2+ -ATPase (PMCA), is uniquely sensitive to oxidation and is progressively lost from specific membrane domains of brain neurons with age. These 'raft' domains create platforms for Ca2+- signaling and are also known to be sites for the processing of the abnormal proteins associated with neurodegenerative diseases. The overall goal of this project is to identify the mechanisms that regulate the localization of PMCA in raft domains and the age-dependent changes underlying the loss of PMCA activity from rafts. Our hypothesis is that this loss is due to enhanced oxidative stress that leads to agedependent changes in the proteins and lipids that regulate the activity and localization of PMCA within rafts. The Aims of this application are to: (1) characterize synaptic membrane rafts from 5, 22 and 34- mos F344/BNF1 rats in terms of protein and lipid oxidation, raft lipid composition, and effects of in vitro oxidative stress; (2) use pharmacological and genetic manipulations to elucidate the role of the raft lipid environment in the membrane localization and kinetic properties of PMCA; (3) determine the role of protein interactions in localization of PMCA in rafts by identifying PMCA binding partners in raft vs non-raft domains, determining their levels in membranes from 5, 22, and 34-mos rats, and altering expression of the major partners in cells to test directly their effects on PMCA localization and activity. Our strategy involves analysis of in vivo brain aging in parallel with in vitro neuronal models for testing specific mechanisms that may explain some of the age-dependent alterations. We make extensive use of expertise in lipidomics, protein identification, and molecular biology available in the Cores, and preliminary data support the feasibility of all aims. Despite the evidence for involvement of rafts in Ca2+ signaling and for Ca2+ dysregulation in agedependent neurodegenerative diseases, nothing is known about aging in neuronal rafts. Our studies will begin to fill that gap and provide new insights into links between aging, altered Ca2+ disposition, oxidative stress and the enhanced vulnerability of the aging brain to the devastating dementias that affect elderly populations. Lav Statement: The proposed studies will enhance our understanding of changes that occur in the aging brain that make it so vulnerable to cognitive impairment and diseases such as Alzheimer's. The ultimate goal is to identify what interventions might slow or prevent some of those changes with advancing age.