Isoform-dependent effects of tau phosphorylation in Alzheimer's disease

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Neuropathology of the microtubule-associated protein tau is central to numerous devastating neurological disorders termed tauopathies, including Alzheimer's disease (AD). Tau promotes microtubule (MT) polymerization and stability via its interaction with tubulin, regulated by phosphorylation to tau in multiple domains. Six major tau isoforms are alternatively spliced in an age-dependent manner. While FTDP-17-causing mutations in tau show isoform-dependent effects on aggregation and microtubule stabilization, there are no tau mutations linked to AD. Meanwhile, isoform-dependent effects of phosphorylation to tau remain unexplored. Interestingly, I discovered that numerous AD-relevant phospho-tau (pTau) sites are also phosphorylated during normal embryonic development, and then decrease over age. Why are specific pTau sites, which are correlated with disease, also expressed during normal development? The parallel between the developmental and pathological states suggests that understanding the function of pTau during development would elucidate the mechanism(s) underlying tau pathology in AD. My preliminary data indicate that i) numerous AD-relevant pTau sites are expressed in normal embryonic mouse brains, ii) pTau is soluble and likely functional in fetal tissue, iii) phosphorylation at sites T231, S235 and S262 impair MT polymerization by the fetal tau isoform but not an adult isoform, and iv) these same pTau sites accelerate aggregation of the adult isoform over the fetal isoform. Recent evidence demonstrates that tau initiates nucleation of MT polymerization. Based on my preliminary data, ! propose that tau phosphorylation has isoform-dependent effects, in which specific pTau sites are detrimental to adult isoforms in an aging brain but functional in the fetal isoform in developing brains. I will explore two aims which will provide critical insight to understanding the effects of tau phosphorylation in each isoform. In Aim 1, I will determine the relationship between the interaction between pTau with both soluble tubulin and stabilized MTs in each isoform. In Aim 2, I will test for the relationship between pTau sites which cause tau to dissociate from MTs with those that accelerate its aggregation and/or seeding activity. Importantly, our lab has uniquely synthesized full-length tau with genuine chemical phosphorylation at sites T231, S235 and S262 for these studies. These experiments will be tested using multiple platforms including these chemically-synthesized proteins, high-resolution single-molecule techniques (single-molecule FRET, fluorescence correlative spectroscopy, and time-lapse TIRF microscopy), and injection of semi-synthesized proteins into mouse brains. The investigator has 6+ years of expertise in working with the biological assays and mouse models described here, and the sponsor and collaborators will guide training in the described chemical and biophysical techniques. I anticipate that my findings will elucidate the function of tau in developing neurons, and will provide insight into mechanisms of pathological tau hyperphosphorylation across numerous tauopathies including AD.