Date of Award

8-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Nutrition Science

Committee Chair

John Burgess

Committee Member 1

Edward L. Bartlett

Committee Member 2

Ignacio Camarillo

Committee Member 3

Kee-Hong Kim

Abstract

Oxidative stress is implicated as a major contributor to the development of diabetes induced peripheral neuropathy. This debilitating condition significantly impacts the quality of life of patients, yet available treatment options are not optimal. They include tricyclic antidepressants, anticonvulsants, serotonin-norepinephrine reuptake inhibitors, selective serotonin reuptake inhibitors, and opiates. Unfortunately, these treatment options only reduce pain by 30-50%, and many patients discontinue use due to side effects. Furthermore, the current treatment options are focused on pain reduction, but not the protection of the peripheral nerves. Since oxidative stress driven by high glucose concentrations has been implicated as the key factor causing peripheral neuropathy, these studies focused on reducing or increasing protection against oxidative stress using dietary compounds with antioxidant properties to ameliorate diabetic peripheral neuropathy. Two dietary compounds with very different mechanisms of antioxidant protection were explored in detail, N-acetylcysteine (NAC) and 3H-1,2-dithiole-3-thione (D3T). To model diabetic peripheral neuropathy, differentiated SH-SY5Y cells were used and stressed with advanced glycation end products (AGE), which form as a result of high glucose concentrations in vivo and cause oxidative stress. In our initial studies, we showed that NAC conferred complete protection against AGE-induced neurite degeneration via a glutathione-mediated mechanism. These studies showed that maintenance of glutathione is critical for neurite structure, as inhibition of glutathione synthesis under non-stressed conditions resulted in significant neurite degeneration. Our next focus was on D3T, a potent nuclear factor (erythroid-derived 2)-like 2 (Nrf2) inducer. D3T generates its antioxidant effect though upregulation of endogenous cellular antioxidant defenses. Previous studies from our lab have shown that D3T treatment paradoxically exacerbates AGE-induced damage, and these prior results were confirmed in the present studies. The mechanism by which D3T potentiates damage was extensively studied. The results of the experiments indicated that D3T potentiates AGE-induced oxidative stress via two critical pathways in the cell-based system used. First, D3T upregulated the Nrf2 responsive gene glucose-6-phosphate dehydrogenase (G6PD), leading to increased G6PD protein expression. Increased expression of G6PD resulted in generation of reducing equivalents that are used by NADPH oxidases to generate superoxide. The oxidative stress damage caused by superoxide generation was then amplified by D3T-mediated reduction of glutathione reductase activity, which resulted in low cellular reduced glutathione concentrations and high oxidized glutathione concentrations. In this manner, D3T inhibited the effectiveness of the endogenous antioxidant defense system and led to disruption of the thiol redox state. In the final set of studies, we found that AGE-induced oxidative stress resulted in a significant increase of protein glutathionylation. Because D3T further disrupts the thiol redox state, it conferred no protection against AGE-induced protein glutathionylation. However, NAC, which was completely protective against AGE-induced neurite degeneration, was also able to fully protect against protein glutathionylation under challenged conditions. Based on the combined results, we conclude that maintenance of the thiol redox state is critical for maintaining neurite morphology, and antioxidants such as NAC that protect the thiol redox state will confer neurite protection in pro-oxidative conditions.

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