Decoding the Mechanostructural Basis and Post-Translational Crosstalk of Citrullinated Tau - General Laboratory Support

Tau pathology develops in many neurodegenerative diseases, known as tauopathies. Cryo-EM has demonstrated disease-specific folds of tau, yet it is unclear how certain conformers arise and become dominant strains. Emerging data implicate less-studied post-translational modifications (PTMs). Citrullination (citR) catalyzed by Ca²?-activated peptidyl arginine deiminases (PADs) is an irreversible modification that neutralizes positive charges on arginine residues, disrupts salt bridges, and cation–π interactions which can stabilize tau structure. We identify tau as a substrate of PADs and found that all 14 arginine’s within 2N4R tau are susceptible to citrullination. We generated site-directed antibodies to citR tau antibodies and found widespread citR tau across nine human tauopathies and in multiple animal models. Functionally, citR impacts tau metabolism, phosphorylation, alters tau aggregation, and impairs cognition in mice. In Alzheimer’s disease tissue, citR-tau correlated with oligomeric tau but not PHF-1, suggesting potentially different pathways across disease stages. We hypothesize that citrullination elicits soluble toxic assemblies that endorse (through irreversible modification) tau proteoforms/ polymorphs and thereby contributes to specific tau architectures that effect functionality. Aim 1 will define PTM crosstalk by quantifying how citR influences tau phosphorylation within the proline-rich, microtubule-binding repeat, and C-terminal domains using CSox-based PhosphoSens. Aim 2 will generated and establish de novo citrullinated tau via Protein Interaction Module-assisted Function X (PIMAX) and through amber codon suppression to measure aggregation kinetics, ultrastructure (TEM), and oligomer profiles. Aim 3 will map single-molecule folding transitions of citR- versus phospho-tau with high-resolution optical tweezers. Collectively, these studies create new tools for citrullinated tau, measure pathogenic PTM crosstalk, define aggregation kinetics, characterize misfolding transitions between phospho vs citrullinated tau. Our work will permit a new area of tau biology that could offer new therapeutic strategies for understudied disease pathways for tauopathies.