AMPA receptors (AMPAR) are a major glutamate receptor in the CNS and are assembled from GLUR1, GLUR2, GLUR3, and GLUR4 subunits. Most AMPARs expressed on hippocampal pyramidal neurons contain the edited form of GLUR2, and are thus impermeable to Ca2? entry. Recent studies have shown that AMPARs play a key role in promoting delayed neuronal death following post-ischemic injury. These glutamatergic receptors undergo a change in subunit composition during post-ischemic reperfusion, changing from a GLUR2-containing Ca2?impermeable AMPAR to a GLUR2-lacking Ca2?permeable receptor. There is substantial evidence that transient global ischemia induced delayed death of hippocampal pyramidal neurons involves activation of these Ca2?permeable AMPARs. At present, the mechanisms responsible for the ischemia/reperfusion-induced expression of GLUR2-lacking AMPARs are not known. However, recent studies indicate that an oxidative stress signaling pathway is responsible for the change in subunit composition of AMPARs. Studies indicate that NADPH oxidase may be the source that initiates the oxidative stress-signaling cascade during post- ischemic reperfusion. The goal of this proposal is to test the hypothesis that increased activity of NADPH oxidase during reperfusion triggers the sequestration and subsequent degradation of the GLUR2 AMPAR subunit leading to an increased surface expression of GLUR2-lacking AMPARs. The proposed study will use acute adult rat hippocampal brain slices. Experiments designed for Specific Aim 1 will test the hypothesis that suppression of NADPH oxidase activity prevents the increase in phosphorylation and subsequent internalization of the GLUR2 AMPAR subunit by ischemia- reperfusion.
In Specific Aim 2, experiments will be performed to test the hypothesis that NADPH oxidase activation accelerates the early endocytic trafficking of GLUR2 AMPAR subunit. Lastly, experiments are designed for Specific Aim 3 to examine whether suppression of NADPH oxidase activity prevents the functional change in AMPARs associated with transient ischemia that is responsible for the delayed death in hippocampal neurons. This proposal seeks to carry out these aims in a manner that emphasizes both graduate and undergraduate training at the College of Health Professions and Biomedical Sciences at The University of Montana. Therefore, this project will incorporate graduate students, Pharm. D. students, as well as undergraduate students. In summary, this project seeks to characterize the oxidative stress-signaling cascade, triggered by increased NADPH oxidase activity that leads to an increased surface expression of Ca2?permeable AMPARs following transient ischemia. As indicated within the proposal description, an emphasis will be placed on student training, including hands-on bench research experiences, seminar and group meeting presentations, as well as eventual dissemination of results in peer-reviewed journals.

Public Health Relevance

A hallmark of post-ischemic reperfusion injury is a change in subunit composition of synaptic AMPARs. This change in AMPAR subunit composition leads to an increase in surface expression of Ca2? permeable AMPARs. These Ca2?permeable AMPARs have been implicated in mediating transient ischemia induced-delayed neuronal death. We propose a novel mechanistic concept in which increased NADPH oxidase activity during post-ischemic reperfusion is the trigger that underlies the change in subunit composition of synaptic AMPARs;changing from Ca2?impermeable to Ca2?permeable AMPARs. We postulate that activation of the superoxide generator NADPH oxidase has a critical role in the synaptic alteration of subunit composition and function of AMPARs in post-ischemic neurons. The proposed studies will test the hypothesis that activation of NADPH oxidase is key to the ischemia- reperfusion induced increase in surface expression of Ca2?permeable AMPARs.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Academic Research Enhancement Awards (AREA) (R15)
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Neural Oxidative Metabolism and Death Study Section (NOMD)
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Bosetti, Francesca
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University of Montana
Other Health Professions
Schools of Pharmacy
United States
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