Project Summary/Abstract Stroke is prevalent in elderly individuals, with greater than 70% of all strokes occurring in individuals above the age of 65. Ischemic stroke, the most common form of stroke, results in delayed neuronal death (DND) in selective vulnerable neuronal populations, such as hippocampal CA1 pyramidal neurons, because of lack of oxygen and nutrients. Recent evidence indicates that glutamatergic AMPA receptors (AMPARs) have a key role in contributing to ischemia/reperfusion (I/R)-induced DND. Following I/R insult, hippocampal GluA2- containing AMPARs are rapidly internalized and degraded. I/R-induced internalization and subsequent degradation of GluA2-containing AMPARs is a key event that underlies the increased synaptic GluA2- lacking Ca2+-permeable AMPARs from either pre-existing extrasynaptic pools and/or mobilization of intracellular pools of these AMPARs to the plasma membrane. We have provided compelling evidence that the superoxide radical has a critical role in the I/R-mediated internalization and degradation of the GluA2 AMPAR subunit. The goal of this proposal is to test the hypothesis that the production of reactive oxygen species following I/R is a key regulator of the phosphorylation, internalization, and degradation of the GluA2 AMPAR subunit. This research will identify the mechanisms responsible for the superoxide- induced trafficking of these AMPAR subunits and identify possible molecular targets to ameliorate the devastating effects of DND following I/R. Experiments designed for Specific Aim 1 will test the hypothesis that activation of ROS generator(s) by I/R promotes the internalization and degradation of GluA2-containing AMPARs in hippocampal slices from aged male and female rats (>18 months old). In Specific Aim 2, experiments will be performed to test the hypothesis that GluA2-containing AMPARs undergo increased phosphorylation in hippocampal slices from aged rats that promotes the I/R-induced internalization and degradation of GluA2-containing AMPARs. Lastly, experiments designed for Specific Aim 3 to test the hypothesis that ubiquitination of GluA2-containing AMPARs in hippocampal slices from aged rats mediates the I/R-induced degradation of the GluA2-containing AMPARs and that ROS generator activation triggers the downstream signaling pathways responsible for AMPAR ubiquitination. To date, few studies have investigated hippocampal AMPAR trafficking in aged rodent models, especially following ischemic/reperfusion injury. This research will be the first to characterize post-translational modifications (phosphorylation and ubiquitination), and trafficking of AMPAR subunits in aged rodent's hippocampus following ischemic/reperfusion injury utilizing a well-established organotypic ex vivo slice model. Findings from this study will provide a framework for future in vivo ischemic/reperfusion injury studies in aged male and female rats (>18 months). This proposal seeks to carry out these aims in a manner that em...