PROJECT SUMMARY/ABSTRACT Hyperphosphorylation to the microtubule (MT) associated protein Tau is the primary driving force of Tau aggregation, a pathological hallmark of Alzheimer’s disease. Tau binds to and stabilizes MTs, especially in neuronal axons. Phosphorylation causes Tau to dissociate from MTs, thus destabilizing them while also increasing the vulnerability of Tau to further phosphorylation and toxic aggregation. Yet, clinical trials aimed at either stabilizing MTs or degrading phospho-Tau (pTau) have yet to succeed. A major caveat of these trials is that they did not stabilize the interactions of specific Tau species to MTs, which is a feasible strategy to stabilizing Tau function and preventing its toxic aggregation. It has been challenging to achieve this clinical goal because there are critical gaps in the Alzheimer’s disease field in distinguishing pathological from functional pTau species, and mapping dysregulation of the pathways that drive Tau toward its hyperphosphorylated state. Most Tau research to date has focused exclusively on the longest Tau isoform even though it is the least abundant isoform in the brain. However, there are a total of 6 Tau isoforms in the brain, expressed at varying protein levels. I have made several discoveries connecting phosphorylation of Tau to its function and dysfunction in an isoform-dependent manner. I found that i) Tau isoforms have different phosphorylation levels in human neurons, ii) pTau species that are associated with Alzheimer’s disease are also abundant and soluble in normal developing brains, iii) phosphorylation regulates Tau-MT interactions and aggregation levels in an isoform- dependent manner. These findings support my central hypothesis that phosphorylation impacts Tau-MT interactions in an isoform-dependent manner (AIM 1), modulated by isoform-specific regulation of Tau-MT interactions (AIM 2). While the study of Tau hyperphosphorylation is a rich field, many studies have been limited by Tau overexpression systems and/or exclusive focus on pathology. Furthermore, high-throughput tools to test for regulators of Tau-MT interactions were not available until recently. I will address these concerns by using new technologies to contrast Tau function and pathology. I will receive training to apply CRISPR prime editing, a new and highly efficient gene editing technique with reduced off-target effects, to endogenously edit phosphorylation sites of interest to Tau to test for isoform-specific effects on its binding to MTs in neurons (AIM 1). Second, I will apply a powerful new technology, pooled optical CRISPR screening, to identify trans-regulators that increase dissociation of Tau from MTs. Optical screening allows for genotype-phenotype matching of virtually any phenotype that can be imaged under a microscope. This aim will uncover both known and novel genetic risk factors for Alzheimer’s disease, thus opening several avenues for my future studies beyond this proposal.