Year of Award

2012

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Neuroscience

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Darrell Jackson

Commitee Members

Howard Beall, Jesse Hay, David Poulsen, J. B. Alexander Ross

Keywords

AMPA Receptor, Ischemia/Reperfusion, NADPH Oxidase, NMDA Receptor

Abstract

Following a stroke, the pathological increase in intracellular calcium arising from the over-activation of post-synaptic glutamatergic receptors has long been established as a key contributor to neuronal death. Also contributing to ischemic/reperfusion-induced neuronal death is oxidative stress, chemically defined as a pathologic state within the cell arising from the increased generation of reactive oxygen and nitrogen species and/or a decrease in the ability of the cell to scavenge and detoxify the reactive intermediates. Strong evidence supports the notion that oxidative stress serves as an important mediator in leading to alterations in intracellular signaling events, ultimately affecting cellular function. However, few studies to date have investigated the role of a key reactive oxygen species generator, NADPH oxidase, in mediating the signaling events leading to alterations in NMDA and AMPA receptor function following ischemia/reperfusion. Therefore, the current series of studies examines the role of reactive oxygen species production from NADPH oxidase following oxygen-glucose deprivation/reperfusion (OGD/R) and the downstream redox-dependent signaling pathways leading to changes in NMDA and AMPA receptor function. In-vitro studies using differentiated SH-SY5Y human neuroblastoma cells were performed to test the hypothesis that NADPH oxidase-dependent ROS production mediates the increased tyrosine phosphorylation status of the NMDA receptor NR2A subunit following OGD/R, ultimately resulting in an enhancement of NMDA receptor mediated cell death. In-situ studies using acute organotypical hippocampal slices prepared from adult male rats were performed to examine the role of the OGD/R-induced increase in NADPH oxidase activity in accelerating the endocytic machineries involved in the surface removal and selective degradation of the AMPA receptor GluR2 subunit following OGD/R. These studies demonstrated that the increased NADPH oxidase-dependent reactive oxygen species production during reperfusion of OGD treated slices was involved in mediating the internalization of the GluR2 subunit, not only through increasing the serine 880 phosphorylation of the GluR2 subunit, thereby leading to a decreased anchoring of GluR2 with membrane scaffolding proteins, but also by enhancing the activity of the endocytic machinery responsible for the internalization of AMPA receptors. Collectively, these studies begin to elucidate a role for NADPH oxidase-dependent signaling pathways in the ischemic/reperfusion-induced pathologic alterations in NMDA and AMPA receptors known to occur following stroke.

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© Copyright 2012 Phillip Howard Beske