Parkinson's disease (PD) is a slow-progressive neurodegenerative disorder affecting 5-6 million people around the globe. The disease is manifested by the rapid deterioration of dopaminergic cells in the substantia nigra portion of the brain; however, the pathological mechanism of selective dopaminergic neuronal death is unknown. A reduction in levels of 3,4-dihydroxyphenylacetaldehyde (DOPAL) is biologically critical as this aldehyde has been shown to be toxic to dopaminergic cells and is a highly reactive electrophile. Investigating neuronal protein targets is essential in determining the cause of toxicity. An essential protein-GAPDH (e.g., glyceraldehyde-3-phosphate dehydrogenase) is an abundantly expressed enzyme known for its glycolytic activity, and recent research has implicated its role in oxidative stress-mediated neuronal death. This work positively shows GAPDH as a target for DOPAL modification, and, for the first time, DOPAL is identified as a potent inhibitor for GAPDH enzymatic activity. LC-MS and other chemical probes (ie. thiol and amine modifiers) show that DOPAL modifies specific –Lys, -Arg, and –Cys residues in the cofactor binding-domain of GAPDH. The enzyme inhibition is also time and DOPAL dose-dependent. DOPAL has a unique structure, containing two reactive functional groups: an aldehyde and catechol ring. In-house syntheses of DOPAL analogues, containing the catechol group and lacking the aldehyde, and vice versa have been tested on GAPDH and do not inhibit or modify GAPDH. Therefore, both the catechol and aldehyde groups of DOPAL are specific to binding with GAPDH and are necessary to achieve modification and enzyme inhibition. In addition to finding a novel enzyme inhibited and modified by DOPAL, this work has also confirmed linking DOPAL levels to a fungicide associated with PD risk. This benzimidazole fungicide, benomyl was shown to inhibit ALDH2 in the SH-SY5Y neuroblastoma cell line via an increase in DOPAL and a decrease in DOPAC. The ratios of DOPAL and DOPAC, the product of ALDH, were measured by HPLC-ECD, and found that benomyl does inhibit ALDH2 in this dopaminergic cell model. The cytotoxicity of benomyl, DA, DOPAL and the combination of DA or DOPAL with benomyl was assessed by MTT assay. Surprisingly, the only toxic combination was the combination of DA or DOPAL with benomyl. In fact, this toxicity appears to be synergistic, as none of the single treatments are significantly toxic to the cells. This synergistic effect also affects GAPDH aggregation. The cell morphology is also drastically different in the presence of the combined treatments, compared to individual treatment of DA, DOPAL or benomyl; cells start to ebb and show apoptotic-like features at just 2h. A second class of pesticides, named chlorpyrifos and chlorpyrifos-oxon were tested for toxicity in PC6-3These compounds were toxic to these cells due to DOPAL accumulation reaching high levels in the 100 µM range. Exposure to environmental toxins such as pesticides and fungicides has long been linked to PD risk, but only recently to DOPAL levels. This work provides a novel mechanism by which fungicide exposure may stimulate PD pathogenesis.

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.