Bcl-2 facilitates recovery from DNA damage after oxidative stress. Oxidative stress is a major factor in affecting the brain during aging and neurodegnerative diseases such as Alzheimer's Disease. Therefore understanding the mechanisms by which neurons can be protected from oxidative stress is critical for the prevention and treatment of such degeneration. Previous studies have shown that bcl-2 gene expression is increased in neurons with DNA damage in AD and the Bcl-2 protein may have an antioxidant effect. The goal of this study was to document the effects of oxidative insults on mitochondrial and nuclear DNA in rat PC12 cells and determine the extent to which Bcl-2 prevents damage or facilitates repair. We used our QPCR gene-specific assay to examine damage in nuclear and mitochondrial DNA. We found that mitochondrial DNA was damaged more extensively than nuclear DNA by either hydrogen peroxide or peroxynitrite. As expected expression of bcl-2 in PC12 cells inhibited peroxynitrite induced cell death. Repair of oxidative damage was accelerated in mtDNA from cells overexpressing bcl-2. These results suggest that bcl-2 up-regulation after DNA damage might facilitate repair after oxidative stress (12). We have tested the hypothesis that inhibition of the electron transport system will lead to the generation of superoxide with the subsequent generation of hydrogen peroxide. These ROS will lead to mtDNA damage and initiate a vicious cycle of damage within the mitochondria. We are testing this hypothesis using several different electron transport inhibitors: 1) 3-nitropropionic acid (3-NPA) which inhibits complex II, succinate dehydrogenase; MPP+ an inhibitor of complex I, NADH dehydrogenase; and rotenone which also inhibits complex I. 3-NPA is a neurotoxin found in some plant (Astragalus) and fungus (Arthrinium) species. It is the suspected toxicant in cases of moldy sugarcane poisoning in rural providence in China. Adminstration of 3-NPA to mice leads to leads to encephalopathy, hind limb weakness, and selective damage to the striatum, hippocampus and thalamus resembling Huntington?s disease. We have been performing experiments with rat PC12 cells which can be differentiated into neuronal cells and express many neuronal specific cell markers. We have also obtained, from Carl Cottman, PC12 cells expressing Bcl-2 (discussed in section 2.1.3.). Treatment of both cell lines with 3-NPA acid resulted in the production of hydrogen peroxide within 15 minutes. Since hydrogen peroxide is freely diffusible, we have measured the amount released into the cculture medium using a horseradish peroxidase assay which oxidizes the fluorophore, Amplex Red in a stoichiometric manner with hydrogen peroxide. The oxidized form of Amplex Red has a 1000-increase in fluorescence intensity. Interestingly, Bcl-2 protein when expressed in PC12 cells decreases the production of hydrogen peroxide by 2-3 fold. As expected 3-NPA leads to a decrease in the mitochondrial membrane potential and a loss of ATP. However, Bcl-2-expressing cells had a much higher membrane potential and higher levels of ATP which while affected by 3-NPA remained higher than the non-treated control PC12 cells. We have measured the production of nuclear and mitochondrial DNA damage in these cells using the QPCR gene-specific assay and found that 4mM 3-NPA produces significant amounts of mtDNA damage within one hour. No nuclear damage could be detected. Bcl-2 expression completely blocked the production of mitochondrial DNA damage after 3-NPA. These results indicate that a complex II inhibitor leads to rapid production of hydrogen peroxide and subsequent mitochondrial DNA damage. Mitochondrial DNA damage proceeds the loss of mitochondrial membrane potential and cell death.Mitochondrial DNA (mtDNA) suffers more damage after oxidative stress than nuclear DNA (nDNA) (Yakes and Van Houten, 1997). In order to understand the molecular events leading to these differences, experiments were performed on telomerase expressing (NHF hTERT) fibroblasts and its normal parental strain (NHF). Using quantitative PCR to examine the formation and repair of H2O2-induced DNA lesions in nuclear and mitochondrial targets, we found that NHF hTERTs show extensive mtDNA damage after exposure to 200mM H2O2 ? which is partially repaired up to 6h. At the same time, the nDNA seems to be resistant to similar stress. Additionally, NHFs, although behaving the same way, show significantly less lesions in the mtDNA than NHF hTERTs. Cell sorting experiments revealed persistent mtDNA damage only in the fraction of cells with low mitochondrial membrane potential (DYm). Further analysis showed also increased production of H2O2 by these cells and apoptotic death. These data suggest a possible mechanism for persistence of lesions in the mtDNA, involving: drop in DYm, compromised protein import, secondary ROS generation and saturated repair capacity. Our findings also point to increased mtDNA sensitivity as a function of hTERT transfection, whilst indicate protection of nuclear genome in comparison to viral immortalization. Mitochondrial studies in the yeast, Saccharomyces cerevisiae. One of the benefits of working in the Laboratory of Molecular Genetics is having the ability to interact with Dr. Mike Resnick?s yeast group. We have developed a synergistic collaboration in which expertise from each laboratory are being used to investigate the effects of mitochondrial damage either through direct chemical insult or through genetic manipulation of strains. One interesting mutant that the Resnick group has developed is a frataxin deletion strain. This gene encodes a protein, frataxin, which is important for transporting iron out of the mitochondria. Low levels of expression of a homologous gene in humans leads to Frederich's Ataxia. We have found that deleting the frataxin gene in yeast leads to an accumulation of DNA damage and a loss of mtDNA.
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