Document Type

Dissertation

Date of Degree

Spring 2016

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Jonathan A. Doorn

Abstract

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.

Public Abstract

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; the pathological mechanism of selective dopaminergic neuronal death is unknown. However, the heightened vulnerability of dopamine neurons is hypothesized to be due to pesticide exposure and the intrinsic presence of dopamine within these neurons. Improper packaging and metabolism of dopamine can be detrimental to these neurons. However, our group and others have shown that a later metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is 1000 times more toxic in vitro and 100 times more toxic in vivo. Therefore, this metabolite is of great interest and its contribution to PD progression. Given the high toxicity and reactivity of DOPAL, it is essential to investigate potential protein targets. This work positively shows that DOPAL targets an essential protein called glyceraldehyde-phosphate dehydrogenase or GAPDH. DOPAL inhibits GAPDH activity and modifies this protein, drastically changing its three-dimensional structure.

This work also provides a mechanistic link between pesticide exposure, DOPAL levels and PD. A fungicide, benomyl was found to be toxic to neurons due to its ability to inhibit breakdown of the enzyme responsible for metabolizing DOPAL, which therefore leads to an increase of DOPAL within neurons. A sub-group of organophosphate pesticides was also tested and found to be toxic to neurons due to DOPAL accumulation.

Keywords

publicabstract, 3, 4-dihydroxyphenylacetaldehyde, DOPAL, GAPDH, Neurotoxicology, Organophosphates and benomyl, Parkinson's Disease

Pages

xv, 106

Bibliography

94-106

Copyright

Copyright 2016 Brigitte Chantal Vanle

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