Date of Award

Fall 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

June Feng

Abstract

Glutathione (GSH) plays an essential role in the intracellular antioxidant defense against the oxidant radicals, especially the ·OH radical. To understand the early and progressive cellular changes in Alzheimer's disease (AD) development, we investigated reduced glutathione/oxidized glutathione (GSH/GSSG) status in a double mutated AD transgenic mouse model (B6.Cg-Tg), which carries Swedish amyloid precursor protein mutation (APPswe) and exon 9 deletion of the PSEN1 gene. Likewise, S-glutathionylation (Pr-SSG) is a specific post-translational modification (PTM) of cysteine residues by the addition of glutathione. S-glutathionylated proteins induced by oxidative stress play an essential role in understanding the pathogenesis of the aging and age related disorder. In addition, 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the major forms of DNA oxidative damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this research was to develop a sensitive method to accurately and rapidly quantify the 8-OHdG by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The method involved the use of specific antibody to detect DNA lesions (8-OHdG) and consecutive fluorescence labeling.

Here, we quantified and compared both GSH/GSSG and mixed-disulfide (Pr-SSG) levels in blood samples and three anatomic positions in the brain (cerebrum, cerebellum, and hippocampus) at 3 age stages (1-, 5-, and 11-month-old) of AD transgenic (Tg)/wild type (WT) mice. The study was designed to characterize and provide insight into the glutathione redox state of both brain tissues and blood samples at different disease stages of this Tg model. The GSH/GSSG ratio in AD-Tg brain tissue started at a higher value at 1-month, fell at the transitional period at 5-month, right before the onset of amyloid plaque, followed by an increase in GSSG and an associated decrease of GSH/GSSG at 11-month. Besides, the high basal levels of Pr-SSG in hippocampus suggest a potential for increased oxidative damage under oxidized conditions and increased GSSG in this vulnerable region.

Moreover, we developed an approach to aid in the early diagnosis of AD by using principal component analysis (PCA) based spectral analysis of oxidized protein electrophoretic profiling. We found that the combination of capillary electrophoresis and PCA analysis of S-glutathionylation distribution characterization can be used in the sample classification and molecular weight (Mw) prediction. PCA can project the S-glutathionyl electrophorectic data into one of six different groups of mice brain and blood samples based on disease progressions and genotype differences. The prediction of the main protein Mw regions that are most susceptible to S-glutathionylation damages in mice brains and blood samples are located at 8.4∼20.3 kDa and 13.2∼37.2 kDa.

Furthermore, the urine sample with 8-OHdG fluorescently labeled along with other constituents was resolved by the capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The low detection limit of 9.0 amol proves the method sensitive enough for the detection and quantification of 8-OHdG in untreated urine samples. The relative standard deviation (RSD) was found to be 11.32% for migration time, and 5.52% for the peak area.

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