The long-term objective of this research is to identify the casual mechanisms associated with paired helical filament (PH) and neurofibrillary tangle formation in Alzheimer's disease. We hypothesize that during recovery neuronal insult and cytoskeletal disruption, tau undergoes changes in conformation, phosphorylation, and cellular localization that resemble tau alterations associated with PHF formation. We further hypothesize that tau associates with a nucleating center, thereby accelerating PHF formation. In preliminary studies, treatment of primary hippocampal neurons of SH- SY5Y neuroblastoma cells with the microtubule depolymerizing agent nocodazone results in tau dephosphorylation and increased perikaryal tau immunoreactivity similar to that observed following neuronal insult. During recovery from nocodazole treatment, tau is phosphorylated at the tau at the tau-12 and PHF-1 epitopes and undergoes changes in conformation similar to those associated with PHFs. These changes are most pronounced in the perinuclear region. In the human brain, PHFs appear to original from a 'nucleating' center or as a perinuclear ring. One candidate for an organizing center is the centrosome, composed of gamma-tubulin and several additional proteins. A second candidate is the plus-end protein kinesin. Kinesin is enriched in the perinuclear region and is known to interact with tau.
Specific Aim 1 will examine the hypothesis that at early stages of NFT formation in the human brain, PHF-tau is associated with organizing or nucleating centers.
This aim will also compare the appearance of PHF-tau is associated with organizing or nucleating centers.
This aim will also compare the appearance of modified tau with the loss or disruption of other microtubule elements.
Aim 2 will examine the hypothesis that microtubule depolymerization and recovery in cultured neurons results in changes in tau conformations, phosphorylation, and cellular localization similar to those associated with NFT/PHF formation.
Aim 3 will examine the association of tau of tau and gamma-tubulin in both the human brain and cultured cells.
Aim 4 will examine the hypothesis that perinuclear changes in tau conformation associated with PHF formation co-localize with kinesin and/or the Golgi apparatus.
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