The long-term objectives of this research are to understand the mechanisms which underlie the cytoskeletal alterations associated with Alzheimer's disease neuropathology. It is increasingly clear that factors other than tau hyperphosphorylation are involved in the formation of neurofibrillary tangles. This application will examine the hypothesis that much of the cytoskeletal disruption evident in neurofibrillary tangles can occur independently of tau hyperphosphorylation. It is proposed that several types of neuronal insult (i.e., metabolic impairment, oxidative stress, sustained depolarization) result in the loss of tau from axons, accumulation in the somatodendritic compartment, and sparing of tau relative to other cytoskeletal proteins (i.e., MAP2, tubulin, neurofilaments). The hyperphosphorylation of tau could be a consequence of its redistribution and occur during recovery from neuronal insult. Experiments proposed in this application will employ primary cultures of rat hippocampal neurons. Results obtained following experimental manipulation of the cultures will expand upon evidence already obtained in vivo. The specific questions which this proposal aims to answer include: 1) Do neuronal insults which enhance perikaryal tau immunostaining also cause a redistribution or loss of other cytoskeletal proteins? Do they alter tau phosphorylation? 2) Does the spatial and temporal pattern of cytoskeletal disruption correlate with alterations in intracellular calcium levels? 3) Are oxidative stress and calpain activation involved in the redistribution and loss of cytoskeletal proteins following neuronal insult? Could calpain substrate specificity account for the sparing of tau relative to other cytoskeletal proteins? 4) Do insults which result in the perikaryal accumulation of tau also impair the ability of tau to bind to microtubules and/or facilitate the aggregation of tau into filamentous structures? 5) Could the hyperphosphorylation of tau occur during recovery from neuronal insult? 6) Does the cytoskeletal disruption observed following neuronal insult in vitro resemble alterations in Alzheimer's disease? Experimental methodologies include primary neuronal culture, immunocytochemistry, immunoblots, confocal and transmission electron microscopy, calcium imaging, and assays for MAP kinase and calpain activity, free radical production, tau phosphorylation, and microtubule binding.
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