Reactive dopamine metabolites and neurotoxicity

Abstract

I would like to thank them for their teaching and reviewing of my data at scientific conferences. A special thanks goes to Dr. Duffel whose lab has worked closely with ours; he always had time to discuss the 'mysteries of enzyme-drug interactions' with me, and the brainstorming of new ideas regarding the anomalies of enzyme assays. I would also like to thank Dr. Quinn for having office hours, and teaching one of the most challenging courses I had taken in graduate school-Mechanisms of Organic Reactions. I would also like to thank Dr. Kevin Rice for his active role in personally recruiting me and the other four classmates of Fall 2010. The idea of successfully finishing graduate school seems inconceivable without the camaraderie and help of my fellow classmates: Eric Rodriguez, Ioana Craciun, Aashay Shah, and Colin Higgins. We were truly a unified class with the same eagerness to learn and to advance science. Special thanks goes to Eric Rodriguez for the countless hours of scientific discussion and for the discovery of new solutions to the small and large problems that arose with instrumentation and cell assays. From over 2000 miles away, I would also like to acknowledge the unwavering and wholehearted support of my family and friends; especially my mother and father, Claudia and Hein, my grandmother Lola, Andrew and Christina. And last but not least, Bella and Zoe for being the best friends a woman could have. v 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 amino acids: Lys, Arg, and modifies Cys and Met residues in the cofactor bindingdomain 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 b...