F2-06-01: major differences between the self-assembly, seeding behavior, and interaction with modulators of heparin-induced versus in-vitro phosphorylated tau

Self-assembly of tau into neurotoxic oligomers and fibrils, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer's disease (AD) and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications or aggregation inducers. The most common induction method is addition of polyanions, such as heparin, yet this artificial system is not an adequate representation of tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in-vitro data that do not represent adequately the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, we compared heparin- induce tau and in-vitro phosphorylated tau aggregation and seeding, and their modulation by a “molecular tweezer”. 2N4R tau was either induced to aggregate by heparin or phosphorylated in-vitro either by ERK2 or by whole rat-brain extract. Aggregation was monitored by thioflavin T fluorescence and electron microscopy. Seeding activity was measured in a FRET-based biosensor cell line. Inhibition of these activities by the molecular tweezer, CLR01, was assessed using the same techniques. Heparin-induced tau behaves differently from in-vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resembles brain-derived tau strains. CLR01 inhibits aggregation and seeding of in-vitro-phosphorylated tau dose-dependently, whereas heparin induction interferes with the interaction between CLR01 and tau. In-vitro phosphorylated tau represents a closer proxy of disease-associated hyperphosphorylated tau than heparin-induced tau and a better system for testing assembly modulators. Using heparin-induced tau aggregation is convenient, but may yield misleading results.