Aging, a major risk factor in Alzheimer's disease (AD), is associated with an increased free radical (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, glutathione (GSH) is the major ROS detoxifying redox buffer within the cell and cysteines are the major reducing buffer in the extracellular matrix. Since the relationship of the prominent ROS damage to aging and AD is unclear, we wanted to know whether A) an oxidative redox shift precedes these markers and leads to macromolecular damage, B) age and AD-related changes can be reversed using redox interventions in neurons and C) modification of the extracellular Cys/CySS redox can change intracellular redox and increase neuron survival. Hippocampal/cortical neurons were isolated across the age-span from non-transgenic (non-Tg) and a triple transgenic Alzheimer's mouse model (3xTg-AD) and cultured in common nutrients to control for age-related hormonal and vascular differences. We found an increase of NAD(P)H levels and redox state in non-Tg neurons until middle age, followed by a decline in old age. The 3xTg-AD neurons maintained much lower resting NAD(P)H and redox state after 4 months, but the NADH regenerating capacity continuously declined with age beginning at 2 months. Compared to non-Tg neurons, 3xTg-AD neurons had lower glutathione (GSH) levels, which preceded age-related increases in ROS levels. The redox deficits in NAD(P)H and GSH were partially reversed using the NADH precursor, nicotinamide. To determine the relative importance of GSH to ROS and cell death, we simultaneously determined GSH depletion and ROS elevation in live neurons across the age-span by titrating with buthionine sulfoximine (BSO), an inhibitor of the rate-limiting enzyme for GSH synthesis, γ-glutamylcysteine synthetase, subunit c (GCLC). We observed that in old age, neuron loss was more dependent on GSH depletion than ROS elevation. Remarkably, the rate of neuron loss with ROS was the same for both genotypes, indicating that cognitive deficits in the AD-model were not caused by ROS. Therefore, we targeted activation of the redox sensitive transcription factor, Nrf2 (Nuclear erythroid-related factor 2) for neuroprotection by 18α-glycyrrhetinic acid to stimulate glutathione synthesis through GCL. . By combining the Nrf2 activator together with nicotinamide, we increased neuron survival against glutathione depletion and beta-amyloid stress in an additive manner. Further, we explored the relative importance of NAD(P)H and GSH to neuron loss in aging and AD. Neurons stressed by either depleting NAD(P)H or GSH indicated that NAD(P)H redox control is upstream of GSH levels and compared to GSH depletion, higher neuron loss was observed with declining NAD(P)H, especially in old age and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyl transferase) and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Lastly, since extracellular redox deficits are seen in aging and AD, and Cys/CySS is the major redox buffer in the extracellular microenvironment, we determined the effects of extracellular redox modification on intracellular redox state, neuron survival and signaling through pAkt/Akt. We found that a reductive shift in extracellular Cys/CySS improved neuron survival, maintained intracellular GSH and NAD(P)H as well as increased pAkt/Akt in aging. Overall, our results strongly support the EORS theory of aging that an oxidative redox shift precedes ROS-mediated damage. A therapeutic reductive redox shift might be used to minimize aging and treat AD
| Identifer | oai:union.ndltd.org:siu.edu/oai:opensiuc.lib.siu.edu:dissertations-1775 |
| Date | 01 December 2013 |
| Creators | Ghosh, Debolina |
| Publisher | OpenSIUC |
| Source Sets | Southern Illinois University Carbondale |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations |
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