Date of Award

12-1-2015

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Pharmacology and Neuroscience

Department

Graduate School of Biomedical Sciences

First Advisor

Thomas Yorio

Second Advisor

Abbot F. Clark

Third Advisor

Meharvan Singh

Abstract

Elevated glutamate produces a cascade of events inducing neuronal death (known as “excitotoxicity”) in which the AMPA receptor (AMPAR), one of the ionotropic glutamate receptors (iGluRs), is hypothesized to play a role in neurodegenerative diseases such as glaucoma. Overstimulation of iGluRs increases intracellular calcium, leading to cellular dysfunction, and eventually apoptotic death of neurons. The activation of AMPARs has been well demonstrated to induce cell death in vivo; however, the mechanism of AMPAR mediated excitotoxicity is not fully understood. We hypothesized that AMPAR desensitization is the determinant of excitotoxicity in retinal ganglion cells (RGCs) in vitro. In this study, we evaluated AMPARs’ dual role in RGCs in mediating both neuroprotection and excitotoxicity following AMPAR stimulation.

Overstimulation of AMPARs (100μM s-AMPA) in purified RGCs was not able to induce the apoptotic pathway or produce RGC death. s-AMPA (desensitizing agonist) was able to increase RGC survival and increase the phosphorylation of cAMP response element-binding protein through the influx of Ca2+. However, RGC survival decreased when RGC cultures were stimulated with kainic acid (non-desensitizing AMPAR agonist) or when co-treated with s-AMPA and cyclothiazide (inhibits desensitization). Following an ischemic-like insult, AMPAR’s alternative spliced flip (decrease desensitization) and flop (increase desensitization) isoforms in purified RGCs were characterized, for the first time, to determine if the flip and flop isoforms play role in RGC excitotoxicity. A decrease in the mRNA expression of GLUA2 and 3 flop isoforms was observed.

In conclusion, we found a dual role for AMPARs in RGCs, where these receptors can mediate both cell survival and cell death. Additionally, for the first time, the decrease in AMPAR desensitization was associated with AMPAR-mediated excitotoxicity, through the changes in the post-transcriptional modifications (alternative splicing and RNA editing of the R/G site) of the AMPAR. This new mechanism of RGC death through AMPAR desensitization gives us a better insight into the pathogenesis and new targets to combat diseases, such as glaucoma, in which neurodegeneration is a hallmark.

Comments

Park, Yong Hwan, The Flip-Flop of AMPA Receptors in Retinal Ganglion Cell Survival. Doctor of Philosophy (Pharmacology and Neuroscience), October 29, 2015,182 pp., 28 illustrations, bibliography, 266 titles. Available world wide December 2016.

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