Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by plaques comprised of Aβ, and neurofibrillary tangles (NFTs) containing the microtubule associated protein tau. Tau pathology closely correlates with neuronal degeneration and cognitive deficits. As the loss of tau protects against Aβ-induced neurotoxicity, tau is thought to act as a pathogenic downstream effector of Aβ to induce neuronal injury. Furthermore, aggregated tau can propagate in a prion-like manner to initiate a self-perpetuating toxic cascade. For these reasons, tau-directed approaches and novel mechanisms of treatment are essential. Hyperphosphorylation of tau is a consistent feature of all tauopathies, suggesting it may be obligatory step in pathogenesis and hence a rational target of modulation. Phosphorylation dissociates tau from microtubules and promotes tau aggregation into paired helical filaments that further accumulate to form NFTs. Tau is natively unfolded, and under normal conditions has little tendency to aggregate; therefore hyperphosphorylated tau in NFTs is a prominent sign of neurodegeneration, and understanding the etiology and pattern of phosphorylated tau is essential. A complication in this analysis is that there are over 80 potential tau phosphorylation sites. Due to the complexity of this problem, tau “hyperphosphorylation” is not precisely defined. Additionally, tau plays important roles during both associative and homeostatic forms of synaptic plasticity, but little is known regarding the phosphorylation events involved in these pathways. Homeostatic responses to hyperexcitation are of particular interest as this type of aberrant overactivity is observed in early stage AD and may be relevant for the initiation of pathogenesis. Here, we will use unbiased mass spectrometry (MS) to perform comprehensive mapping of tau phosphorylation patterns during specific physiological conditions as well as in disease models. Compiling phosphomaps into an “atlas” of tau modifications will begin to decipher the phosphorylation code that governs tau function in physiology and pathology. In Aim 1, we will use qualitative and quantitative MS approaches to define tau phosphorylation patterns during different forms of synaptic plasticity in cultured hippocampal neurons, examining a time course after each paradigm to observe time-dependent changes. We will also use specific kinase inhibitors with the above stimulation protocols to facilitate identification of endogenous tau kinases and signaling pathways. In Aim 2, we will use MS to identify tau phosphorylation patterns in a humanized double knock-in model of familial AD. As a primary tauopathy model of frontotemporal dementia, we will use P301L-tau in the background of humanized tau knock-in mice. We will examine different ages to understand the earliest tau modifications and profile of disease progression, as well as male vs. female mice to elucidate sex differences in disease severity. Defining the physiolog...