Abstract
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that begins decades before the onset of clinical symptoms of dementia. AD can be distinguished from other neurodegenerative diseases by its pathological hallmarks which are plaques and intracellular neurofibrillary tangles in the brain. The plaques mainly comprise of amyloid-beta (Aβ) peptides while the tangles are primarily composed of a microtubule associated protein called tau. Human central nervous system contains six isoforms of tau. Each isoforms contains 80 potential phosphorylatable sites and ~50 have shown to be phosphorylated. Among those ~50, 3 sites, threonine 181 (T181), threonine 217 (T217) and threonine 231 (T231), were recently found to have high predictive power for early AD symptom onset many years later. However, the functional role of these three phosphorylated sites have been understudied. Among the three phospho-tau variants, tau phosphorylation at T217 (taupT217) is emerging as a superior predictor of symptom onset as tau positron emission tomography (PET) but little is known about its cellular localization and function in AD pathogenesis. TaupT217 can also be used as a potential therapeutic target if its mechanism of action is properly understood. Thus, understanding the cellular distribution and functional characteristics of taupT217 was the main focus of this dissertation.
To determine the cellular and intracellular deposition, human brain and cultured mouse neurons were analyzed by immunoblotting and immunofluorescence for total tau, taupT217, taupT181, taupT231 and taupS396/pS404. Direct stochastic optical reconstruction microscopy (dSTORM) was used to localize taupT217 in cultured neurons. To determine binding affinity of tau phosphorylation at T217 with microtubule and its effect on neurotransmission, tau tagged with enhanced green fluorescent protein was expressed in fibroblasts and in cultured neurons. I used fluorescence recovery after photobleaching (FRAP) to report tau turnover rates on microtubules and calcium imaging to report synaptic function by stimulating or inhibiting AMPA and NMDA receptors.
In this dissertation, I show evidence that in the brain, taupT217 appears in neurons at Braak stages I-II, becomes more prevalent later and co-localizes partially with taupT181 but not with taupT231. In cultured neurons, taupT217, is increased by extracellular tau oligomers (xcTauOs), is associated with developing post-synaptic sites, and mediates AMPA and NMDA receptors. Meanwhile, in fibroblasts, FRAP recovery was faster for EGFP-tauT217E than EGFP-tauWT.
The results I have gathered imply that taupT217 increases in the brain as AD progresses and is induced by xcTauOs which may act as seeds in inducing intracellular taupT217 level in AD neurons. Post-synaptic taupT217 suggests a role for T217 phosphorylation in synapse impairment by mediating AMPA and NMDA receptors dependent neurotransmission. My results also imply that reduced microtubule affinity of tau phosphorylated at T217 causes dysregulated fast axonal transport.
This dissertation is an initial step towards understanding early AD therapeutics targeting phospho-tau epitopes. In the future, such strategy could potentially stop or slow AD progression in its early stages itself, rather than later stages.
