In order for the neuronal cytoskeleton to function properly, intracellular calcium levels and oxidative states must by maintained within specific limits. Disruptions in the calcium homeostatic mechanisms, or conditions of heightened oxidative stress are likely to contribute tot he pathological dysfunction of the cytoskeleton in certain neurodegenerative diseases, especially Alzheimer's disease (AD). These abnormal conditions have been suggested to result in the disregulation of the activities of specific enzymes, including specific protein phosphatases and kinases, which ultimately contribute to abnormal cytoskeletal rearrangements and the death of the neuron. The microtubule-associated protein tau is a critically important phosphorylated cytoskeletal protein necessary for the maintenance of neuronal structure and function. Tau, in a hyperphosphorylated form, is also the predominant protein of paired helical filaments (PHFs) which are found in Alzheimer brain. The overall goals of this proposal are to examine the modulation of the phosphorylation state of tau by calcium- dependent processes, and to determine the role of oxidative/reductive conditions in mediating the metabolism and function of tau. Our overall hypothesis is that oxidative stress and disrupted calcium homeostasis contribute to the pathology of AD by altering the activities of specific enzymes which leads to modifications of tau which are pathological. The goals of this proposal are to test the following hypotheses: (1) During programmed cell death, specific calcium-mediated alterations occur in the phosphorylation state of tau as a part of the cascade leading to cell death, (2) heightened oxidative stress results in alterations in the phosphorylation state of tau, (3) compromised mitochondrial function, which produces an increase in intracellular calcium levels, results in alterations in the phosphorylation state of tau, (4) after transient calcium-stimulated dephosphorylation, tau becomes more extensively hyperphosphorylated and (5) specific mechanisms eliciting increases in intracellular calcium and heightened oxidative stress result in calcium- mediated alterations in the localization of tau within the cell. In preliminary studies, treatment of NGF-differentiated PC12 cells with low levels of the calcium ionophore, A23187 resulted in an increase ina the phosphorylation of tau, while increasing oxidative stress by depleting reduced glutathione resulted in a dephosphorylation of tau. Additionally, treatment of rat brain cortical slices with an inhibitor of oxidative phosphorylation or the excitatory amino acid, N-methyl-D-aspartate resulted in a site-selective dephosphorylation of tau by the calcium/calmodulin- dependent phosphatase, calcineurin (phosphatase 2B). These and other preliminary findings clearly demonstrate that the studies outlined in this proposal will yield important new information on how the phosphorylation state of tau is modulated in response to calcium and oxidative stress, and the functional consequences of these alterations.
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