DOI

10.17077/etd.3ppza3z6

Document Type

Dissertation

Date of Degree

Summer 2017

Access Restrictions

Access restricted until 08/31/2020

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Doorn, Jonathan A.

First Committee Member

Roman, David L.

Second Committee Member

Duffel, Michael W.

Third Committee Member

Spies, Michael A.

Fourth Committee Member

Narayanan, Nandakumar

Abstract

Parkinson’s disease (PD) is a chronic and progressive movement disorder affecting an individual’s ability to move, and can become life threatening when it progresses to the point where an individual has difficulties swallowing, breathing, and chewing. PD is a neurodegenerative disorder caused by the damage of neurons, leading to the loss of nerve function and structure in the brain. Specifically, PD is characterized by the selective loss of the substania nigra, the dopamine (DA)-containing region of the brain. Due to loss of DAergic neurons, it has been suggested that DA serves as an endogenous toxin when there are alterations in the synthesis, metabolism, and regulation of DA. The pathogenesis of PD remains unclear, and many are working on determining what factors cause this neuronal death. Factors hypothesized to be important include: aging, genetics, endogenous toxins, and environmental toxicants.

The aim of this work is to explore the role of endogenous neurotoxins, such as toxic dopamine metabolites, oxidative stress (OxS), and reactive oxygen species as contributors to the neurotoxicity relevant to PD, and to examine the potential for regulation of this toxicity by alterations in the antioxidant status of the cell. DA can undergo metabolism by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a highly toxic and reactive metabolite; that is hypothesized as a contributor to the neurotoxicity observed in PD. Subsequently, DOPAL can be further metabolized by aldehyde dehydrogenases or reductases to form 3,4- dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), respectively. When evaluating all of these metabolites, DOPAL displays the greatest toxicity both in vitro and in vivo. DOPAL contributes to cell toxicity through a variety of mechanisms; these include: 1) it is able to react with proteins, leading to covalent modification at Lys and Arg residues causing the formation of adducts 2) DOPAL can autooxidize to form quinone species, which are reactive with proteins 3) autooxidation and protein modification by DOPAL results in the generation of reactive oxygen species (ROS) (H₂O₂, O₂•−), which are also toxic. Of note, increasing ROS can impact the OxS levels, creating an imbalance that contributes to cell damage. This insult can include inhibiting the carbonyl metabolizing enzymes, further increasing DOPAL levels. During these interactions, damage occurs to proteins, enzymes, and DNA, causing an inability for the cell to perform properly, consequently leading to cell death.

The initial work, described in Chapter 3, was determination of ROS and secondary insults that are produced during DOPAL-mediated neurotoxicity. Methodologies utilizing fluorescence detection were able to identify the production of both hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂•−). The formation of these ROS can result in an imbalance in oxidative status, contributing to augmented OxS in the cell. These ROS were produced both in purified protein assays, as well as, in cell based studies. These assays investigated formation of ROS during protein interaction, but were also tested in the presence of known toxins that have been correlated with PD.

The work described in Chapter 4 explores conditions in these neurons that can impact the alteration of OxS through ROS. It was hypothesized that oxygen presence is necessary to catalyze the reaction of DOPAL with proteins. Therefore, work was completed to discover if oxygen deficiency could regulate DOPAL-protein interactions. Identification of protein modification, following oxygen eradication, confirmed that inhibition of DOPAL’s reactivity towards proteins succeeds the loss of oxygen. This led efforts to focus on other mechanisms by which to alter cellular oxidative status to influence DOPAL’s function in these cells. Additional work was completed to discover if radical scavengers similarly control resultant toxicity from DOPAL activity. As previously published, radical scavengers, such as tricine, exhibit a protective effect in regards to modification of proteins. Furthermore, we believe that oxidative status can serve as a target for mediation of DOPAL neurotoxicity.

If affecting the capability of producing ROS species can impact OxS in DOPAL-mediated toxicity, it is believed that utilizing agents, such as antioxidants, can serve as a new potential treatment for PD. Chapters 5 (cellular models) and 6 (in vivo model) explore efforts to alter (+/-) antioxidant levels via addition of N-acetylcysteine (NAC), diamide (Dia), and buthionine sulfoxide (BSO). It was found that antioxidants, such as NAC, attenuate the adduction of proteins by DOPAL, alter DA metabolite levels, and inhibit behavioral characteristics of PD in the in vivo model. Conversely, oxidants Dia and BSO increased DOPAL and its subsequent modification of proteins.

Finally, Chapter 7 includes a conclusion of the work documented here and addresses future potential directions. This project includes so new findings that need to be further characterized resulting in many future direction that can be explored. One major direction in which this project can be taken would be further validation of NAC to serve as a novel therapy for PD. The future directions will include all aspects of this project including a brief discussion of examining NAC analogs to increase bioavailability leading to a more potent drug model.

To date there are limited answers into what is causing this neurodegeneration, and currently, there is no cure for PD. Therefore, my thesis research is making an impact in the field as it, has explored the ways in which a known toxic metabolite is leading to death of these neurons, has identified secondary products that are contributing to the toxicity observed, and has developed a potential new therapy for PD utilizing antioxidants. All of these will help advance research in the field to continue to identify new targets in this cellular pathway leading to a better understanding of the cause of PD.

Keywords

3, 4-dihydroxyphenylacetaldehyde, Antioxidants, Dopamine, Oxidative Stress, Parkinson's Disease

Pages

xviii, 149 pages

Bibliography

Includes bibliographical references (pages 135-149).

Copyright

Copyright © 2017 Josephine Helen Schamp

Available for download on Monday, August 31, 2020

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