Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule regulation

Abstract

Tauopathies such as Alzheimers disease are characterized by the aggregation and increased phosphorylation of the microtubule-associated protein tau. The pathological changes in tau are closely linked to neurodegeneration, making tau a prime candidate for intervention. However, the multiple facets of tau function and the lack of cellular tauopathy models that could support mechanism-based drug development hampers progress. Here we report the development of a live-cell imaging approach to quantitatively monitor pathological changes of human tau as it interacts with axonal microtubules. We show that a full-length aggregation-prone tau construct exhibits reduced interaction with microtubules as it increasingly aggregates. Through chemoinformatic analyses, we identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that inhibit tau aggregation and modulate tau phosphorylation. We found that PHOX15 restores the physiological microtubule interaction of aggregation-prone tau in neurons and inhibits the first phase of tau aggregation in vitro. Furthermore, we report that PHOX15 inhibits the tau kinases GSK3beta and Cdk5, alters the kinome activity of model neurons, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and indicate that the binding of PHOX15 in one of the channels reduces the protofilaments ability to adopt a PHF-like conformation. The data show that our imaging approach provides a useful tool for identifying compounds that modulate tau-microtubule interaction in axons. We demonstrate that a polypharmacological approach to simultaneously treat tau aggregation and tau phosphorylation is able to restore physiological microtubule regulation, identifying PHOX15 as a promising drug candidate to counteract tau-induced neurodegeneration.

Redox signaling by hydrogen peroxide modulates axonal microtubule organization and induces a specific phosphorylation signature of microtubule proteins distinct from distress

“…PC12 cells were neuronally differentiated by culture in serum-reduced DMEM with 100 ng/ml 7 S mouse NGF for 4 days and treated with 150 µM H2O2 for 3 hours, 0.5 mM arsenite for 20 min, or left as respective controls. Proteome and phosphoproteome analysis were essentially performed as described previously (Pinzi et al, 2024). Briefly, cells were incubated in lysis buffer (8M urea in 50mM Tris/HCl, pH 7.8) supplemented with Phos-Stop tablets (Roche Diagnostics GmbH, Germany), sonicated and cleared by centrifugation.…”