Both oxidative damage to proteins and cytoskeletal abnormalities have been detected in Alzheimer's disease. In neurons containing neurofibrillary tangles, a lesion characteristic of Alzheimer's disease, the number of microtubules is reduced by 50%. Furthermore, it is very likely that the reactive species peroxynitrite anion and hypochlorous acid are formed in neurons that contain neurofibrillary tangles because markers of oxidative damage including nitrotyrosine and 3-chlorotyrosine are present. Our in vitro work with microtubule proteins including tubulin, tau and microtubule- associated protein-2 shows that cysteine oxidation by peroxynitrite anion correlates with a loss of function. Thus, microtubule proteins may be more susceptible to oxidative damage by peroxynitrite anion and other reactive oxygen species because of the greater reactivity of their cysteine thiols.
The specific aims of this proposal are: 1) to study the effects of nitric oxide and nitroxyl donors on microtubule protein cysteines; 2) to assess the ability of ascorbic acid to reduce protein and small molecule disulfides; 3) to study the oxidation of microtubule proteins by hypochlorous acid and chloramines and 4) to use biotin- and fluorescein-labeled glutathione analogues to study microtubule protein S-glutathionylation. Studies examining the effects of cellular oxidants on microtubule proteins are essential because they provide a starting point for understanding how reactive species including peroxynitrite anion, nitric oxide, hypochlorous acid and chloramines could jeopardize cell viability in vivo. It has been shown conclusively that these oxidants are produced in neurons and that there is a positive correlation between increased oxidation and aging. Cytoskeletal proteins like tubulin and microtubule-associated proteins are among the most abundant proteins in neurons; thus, they are likely targets for modification by oxidants. In addition to our interest in the potential for cellular damage by oxidants, we are intrigued by the possibility of redox regulation of microtubule dynamics in vivo. Strong evidence links increased oxidative damage with the development of neurodegenerative diseases including Alzheimer's and Parkinson's disease. Reactive molecules, derived from the oxygen we breathe, cause reversible and irreversible oxidative damage to biological macromolecules including proteins, lipids and DNA. Researchers must consider and assess the effects of reactive molecules on abundant brain proteins because it is very likely that many proteins are damaged as we age. ? ?
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