Development and implementation of new techniques to study biomarkers in Huntington disease
Angle, Erika Natalie Ebbel
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Huntington disease (HD) is a neurodegenerative disease characterized by motor dysfunction and emotional disturbances. No current therapy delays onset or slows progression. One challenge to developing therapies is that the molecular mechanisms of the disease are not fully understood. Identification of small molecule biomarkers defining "disease state" would assist in elucidating the causes of changes occurring throughout disease progression and in developing therapeutics to address these changes. The thesis research had three aims. The first aim was to develop a method employing offline liquid chromatography-electrochemical (LC-EC) array and parallel LC-EC array-mass spectrometry (MS) to identify small molecules in a sample set of HD and control subjects for which we had found several statistically significant but qualitatively unidentified compounds using offline LC-EC array. The second aim was to apply these technologies to identify biomarkers of HD progression; the third was to determine whether the candidate biomarker(s) interact with plasma proteins because of the possibility that small molecule covalent binding affects protein aggregation, the putative cause of HD pathology. A method was developed to use offline and parallel LC-EC arrays to identify metabolites in an HD drug trial of phenylbutyrate. The method was then applied in a pilot study of plasma from HD patients. The analyses yielded the unexpected result of discovering several new metabolites of the anti-inflammatory drug Etodolac™. After method modification, a larger study of HD was undertaken to monitor HD progression and to search for disease progression biomarkers amongst groups of HD patients stratified by stage of disease. A potential biomarker for HD, indole-3-propionic acid, was identified. A method was developed to covalently link small molecules and peptides or proteins with offline electrochemical cells and allowed determining the potential binding site of 5-hydroxytryptophan, a metabolite selected as a model of covalent binding that is of interest because of a known neurotoxic role of oxidized 5-HTP. The biochemical localization of indole-3-propionic acid and potential mechanisms for its decrease in HD patient plasma were investigated. In summary, technologies for investigating small molecule-protein interactions were developed and applied to HD, and suggested new approaches to discovery of disease biomarkers and mechanisms.
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