Molecular recognition of pathological tau fibril conformations

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ABSTRACT Tau is an intrinsically disordered protein that binds microtubules in healthy neurons but forms filamentous aggregates that drive neurodegenerative diseases known as tauopathies. Cryo-EM structures from patient samples revealed that these amyloid fibrils adopt unique tauopathy-dependent conformations that differ from those of heparin-induced recombinant tau fibrils in vitro. These observations suggest that tau fibril morphology begets disease phenotype, and that these aggregates propagate from neuron to neuron, recruiting tau monomer in a “prion-like” mechanism. However, the mechanistic basis for transformation of the healthy intracellular tau pool to pathological aggregation remains largely undefined, and there are no in vitro methods for generating tauopathy-like fibril conformations with recombinant tau. Recently characterized alternative inducers of tau fibrillization in vitro may faithfully recapitulate tauopathy-associated fibril morphologies; however, an understanding of the cellular and environmental factors driving conformation-specific fibrillization is hampered by the lack of tools for readily determining fibril conformation and the incomplete understanding of molecular crosstalk with tau posttranslational modifications (PTMs). Here, I propose to leverage two unique molecular recognition platforms to develop conformationally selective tools for rapid identification of tau fibril conformations. My hypothesis is that cellular factors modulate the tau conformational landscape to promote amyloid fibril formation in neurodegenerative disease and that new tools for detecting and inhibiting this process will be useful therapeutics. For my first aim, I will utilize a small molecule fluorogenic probe library to identify molecules that recognize disease-associated fibril conformations by comparing dye binding profiles of Alzheimer’s disease patient-derived fibrils and fibrils generated in vitro with diverse polyanionic inducers. For my second aim, I will use a yeast-displayed humanized nanobody library system to identify tau conformation-selective nanobodies that will and serve as candidates for immunotherapeutics that inhibit the addition of monomers to tau fibrils and expand and multiplex molecular recognition of fibrils. For my third aim, I will use chemical biology approaches to reconstitute disease-associated tau ubiquitinations and, in combination with newly identified, conformationally unique in vitro-induced tau fibrils, systematically examine the effect of ubiquitination on fibrillization propensity, kinetics, and conformation and changes in fibril protein-protein interaction networks. Upon the successful completion of this proposal, I envision a new toolkit for neurodegenerative disease precision medicine in which small molecule fluorogenic probes detect tau fibril conformation/PTM status, and then fibril propagation is specifically inhibited using nanobody immunotherapeutics.