Major Differences between the Self-Assembly and Seeding Behavior of Heparin-Induced and in Vitro Phosphorylated Tau and Their Modulation by Potential Inhibitors

Abstract

Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer’s disease 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 (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced (28784767Proc. Natl. Acad. Sci. U.S.A.201711490809085). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure–activity relationship study demonstrates that 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 resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau.

The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice

“…Membrane-bound fractions were not used because the detergent causes cytotoxicity. The measurements were performed as described previously (26). Transduction complexes were made by combining 8.75 µL Opti-MEM (Gibco) and 1.25 µL…”

“…The cells then were incubated with the transduction complexes for 48 h and subsequently visualized using uorescence microscopy. The integrated FRET density was analyzed by ow cytometry as described previously (26,32).…”

“…The detergent present in the membrane-bound fraction is toxic to the cells, preventing the use of this fraction in the assay. In each experiment, the cells were imaged by uorescence microscopy using the uorescence of YFP to obtain a qualitative assessment of the seeding response and the number of seeds was quanti ed by measuring the FRET signal between CFP and YFP using ow cytometry, as described previously (26).…”

“…CLR01 also inhibited seeded, intracellular tau aggregation in a biosensor cell line (26). The main binding region for CLR01 on tau was identi ed by NMR (26) and mass-spectrometry (27) to be the repeat domain, which forms the core of the aggregates.…”

“…More recently, CLR01 was found to inhibit the aggregation of the longest form of tau, 2N4R, either when it was induced to aggregate by heparin or when the protein was phosphorylated in vitro rendering it prone to aggregation (26). CLR01 also inhibited seeded, intracellular tau aggregation in a biosensor cell line (26). The main binding region for CLR01 on tau was identi ed by NMR (26) and mass-spectrometry (27) to be the repeat domain, which forms the core of the aggregates.…”