We are testing the hypothesis that the accumulation of oxidative DNA damage contributes to the neuronal dysfunction seen in neurodegenerative diseases by utilizing several biological models like transgenic mice, patients post-mortem tissue and cultured lymphoblasts from patients with neurodegenerative diseases. We are now focusing on Alzheimer's disease (AD) since this is the most prevalent form of dementia in people 65 years or older. Using in vitro assays and DNA substrates containing single oxidized lesions we investigate whether the activities of the enzymes involved in repair of oxidative DNA damage are altered in AD. Oxidative DNA damage is mainly repaired by the base excision repair (BER) pathway, and we are focusing on the enzyme components of this pathway. In cultured AD fibroblasts we found that exogenously-generated oxidative DNA lesions are repaired as efficiently in AD as in controls, which suggests that alterations in oxidative damage processing may be highly cell type-specific. We next measured BER capacity in tissue extracts obtained from well-established animal models for AD. We have used three transgenic mouse models, expressing mutant human amyloid precursor protein 1 (APP1) gene;a double transgenic mouse expressing mutant APP1 plus mutant presenilin 1;and a triple transgenic mouse expressing the two previous genes plus a mutated form of Tau. All these gene products are involved in the formation of plaques and tangles in the AD brain and these mice develop several AD-like symptoms in an age-associated fashion. Thus, we compared DNA repair activities in mice before and after the onset of the disease. Moreover, because some regions of the brain are pathologically affected (for example corpus callosum and hippocampus atrophy) while other regions seem to remain unaffected, we measured DNA repair capacity in extracts from 5 different brain regions in normal and AD-model mice. We also followed age-associated changes in DNA repair capacity in these regions in wild type mice. Our results show that BER activities in mitochondria varied greatly among striatum, frontal cortex, cerebellum, hippocampus and brain steam, with brain steam having highest and striatum the lowest DNA glycosylase activities. We observed a general decrease in BER efficiency in brain with age;however the age-associated changes also differ among the regions. In contrast, we observed decreased activity for some BER enzymes, but not all, and this was restricted to two regions of the brains of older AD mice when compared with young, pre-symptomatic mice. The regions with altered BER activity did not correlate with the pathologically affected ones and we are now investigating whether this is due to cell type-specific sensitivity to environmental factors. Nonetheless, mice do not reflect all the pathological hallmarks of AD in humans, thus we measured BER activities in post-mortem tissue samples from AD patients and age-matched cognitive normal controls. We found a significant decrease in BER activities in samples from AD patients, when compared to age-matched controls. Both activity and protein levels of two core enzymes of the BER pathway, uracil DNA glycosylase (UDG) and polymerase beta, were altered in the brains of the AD patients. Moreover, we found similar decreases in BER activities in samples from patients suffering from Mild Cognitive Impairment (MCI), which is considered a pre-Alzheimers state. In these patients we found an inverse correlation between BER activity and Braak stage. The Braak stage is a measurement of the number of plaques and tangles and is considered a surrogate index for the pathology. Lower BER activities were observed both in cortex and cerebellum samples, indicating that AD-associated neuronal cell death could not account for the differences. Together our results suggest that lower BER may be a predisposing condition in the development of the AD pathology. Mitochondria likely contribute to AD pathology and BER DNA repair in mitochondria from AD patient samples has now been evaluated. We find that 5-hydroxyuracil incision and ligase activity are significantly diminished in mitochondrial protein extracts derived from mitochondrial extracts of post-mortem AD brain samples relative to controls. Oxidative damage is one of the earliest markers of AD and our results expand the characterization of deficient BER in AD brain samples. Together, our work and others, suggest that deficiencies in BER enzymes might contribute to the accumulation of oxidative damage in both the nuclear and mitochondria of AD patients. We are continuing to pursue the notion that oxidative DNA processing is deficient in AD and that this may lead to pathological changes. To define what kind of changes occurred in AD cells, we recently performed gene expression analysis on a set of AD fibroblast cells and compared them with matched normal cells either untreated or treated with hydrogen peroxide or menadione. Both of these agents induce oxidative damage and produced a similar pattern of gene expression changes. When compared to either untreated or treated normal cells, the AD samples gene expression pattern resembled that of oxidatively stress normal cells. We also demonstrated that the AD patient samples had more oxidative damage than the controls. We proposed, in part, that the gene expression pattern changes seen in AD cells may arise from the elevated oxidative stress experience by the AD cells. Thus, we are continuing to test the hypothesis that lower BER may predispose individuals to development of AD pathology.
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