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 prevalent neurodegenerative disorder which affects over a million people in the United States. This disease is marked by the selective loss of dopaminergic neurons in the substantia nigra, leading to a decrease in the important neurotransmitter dopamine (DA), which is essential for the initiation and execution of coordinated movement. Currently, the pathogenesis behind PD is unknown, but there is evidence that both exogenous causes, such as pesticides and metals, as well as endogenous causes, such as reactive oxygen species or reactive metabolism intermediates, may play a role in the onset and progression of the disease. DA is catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized by aldehyde dehydrogenase and aldehyde reductase to the acid and alcohol products, respectively. Studies have demonstrated the reactivity of DOPAL with peptides and proteins, leading to covalent modification which may be detrimental to protein action. Furthermore, studies have shown that DOPAL is toxic, leading to a decrease in cell viability. Due to this, it was of interest to further study DOPAL and how it may play a role in the onset and progression of PD. It was of particular interest to determine protein targets of DOPAL modification. Until recently, no protein targets were identified and the cellular consequence of elevated DOPAL had not been fully studied. It has been previously shown that the important enzyme, tyrosine hydroxylase (TH) is inhibited by other catechols, including DA. This enzyme catalyzes the rate-limiting step in DA synthesis, oxidizing tyrosine to L-DOPA which is further metabolized to DA. Therefore, it was of interest to determine the effect of DOPAL on TH activity. It was hypothesized that DOPAL modifies and inhibits TH, leading to a decrease in the production of L-DOPA and DA. This work employed the use of a dopaminergic cell model (PC6-3 cells), to positively identify TH as a protein target of DOPAL modification. It also used both cell lysate as well as PC6-3 cell studies to investigate the effect of DOPAL modification on TH activity. Mass spectrometry was also utilized to determine sites of protein modification on TH. Results show that TH is potently inhibited by DOPAL modification, leading to a significant decrease in both L-DOPA and DA. Furthermore, DOPAL inhibition appears to be slowly-irreversible, with enzyme activity showing a time- and concentration dependent in recovery after preincubation with DOPAL. A novel cloning and purification procedure was used to clone human recombinant TH, which was used in mass spectrometry studies in which five sites of DOPAL modification were discovered. Furthermore, a real-time assay for TH activity was developed using a plate reader to spectrophotometrically observe the formation of L-DOPA over time. These data demonstrate the toxicity and potent enzyme inhibition by DOPAL and implicate DOPAL as a neurotoxin relevant in the pathogenesis of PD.