Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Tonia S. Rex, Ph.D.

Committee

Edward Chaum, M.D., Ph.D. Eldon E. Geisert, Ph.D. Ramareddy V. Guntaka, Ph.D Michael A. Whitt, Ph.D.

Abstract

Neuroprotection is a strategy to prevent or reduce neuronal cell death regardless of the underlying cause. Erythropoietin (EPO) exhibits neuroprotective effects in a variety of in vitro and in vivo models of neuronal cell death although its classical function is the regulation of red blood cell production. EPO crosses the blood brain barrier and therefore can be delivered systemically. However, long term treatment with exogenous EPO causes over stimulation of the erythropoietic pathway which can be lethal. The erythropoietic and neuroprotective pathways are regulated by different receptors; further, it is possible to modify EPO to primarily, if not solely, activate the receptor responsible for neuroprotection. An example of this is a novel form of EPO that we developed by altering a single amino acid, arginine to glutamate, at position 76 (EPO‑R76E). The present study uses recombinant adeno-associated virus (rAAV) gene therapy and systemic delivery of EPO‑R76E to assess treatment potential for degenerating neurons in an induced model of neurodegeneration and a spontaneous model of glaucoma.

Optic nerve crush is a model of retinal ganglion cell (RGC) death. Crushing the optic nerve causes the axons to undergo Wallerian degeneration and apoptotic loss of retinal ganglion cells over time. This pattern of cell death is very similar to that observed in glaucoma. The BALB/cByJ mouse strain is highly susceptible to optic nerve crush and therefore was selected to examine the potential effects of EPO‑R76E as a neuroprotectant. Mice received a single intramuscular injection of rAAV carrying either Epo or EpoR76E. Upon reaching peak gene expression (30 days), mice were subjected to optic nerve crush. Neuronal cell death was examined in both the ganglion cell layer and the optic nerve. Treatment with either Epo vector resulted in a significantly greater amount of cells at the ganglion cell layer compared to crushed controls but had no effect on axon number. The observed protection occurred in a dose dependent manner. Furthermore, EPO‑R76E did not stimulate erythropoiesis to the same extent as wild type EPO.

The neuroprotective effects of EPO‑R76E following optic nerve crush prompted its investigation as a treatment for glaucoma. The DBA/2J mouse strain develops a naturally occurring pigmentary glaucoma as a result of increased intraocular pressure (IOP). At 1 month of age DBA/2J mice received a single intramuscular injection of rAAV carrying either Epo or EpoR76E. The mice were aged to 10 months and IOP was used as a marker for glaucoma. Treatment with either Epo vector prevented death of the RGC somata and their axons within the optic nerve. Furthermore this morphological rescue was accompanied by preservation of visual function as demonstrated by flash visual evoked potentials. The protection provided by EPO‑R76E was independent of IOP and did not cause a significant increase in hematocrit. This is the first example of a neuroprotective agent for glaucoma that protects the RGC soma as well as the axon while maintaining function.

DOI

10.21007/etd.cghs.2011.0389

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