Photo-Excited Toluidine Blue Disaggregates the Repeat Tau and Modulates End-Binding Protein EB1, Cytoskeletal Structure in Neuronal Cells

Abstract

Alzheimer's disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. In the present work, we studied the effect of Toluidine Blue and photo-excited Toluidine Blue on the aggregation of repeat Tau using in vitro assays. The in vitro experiments were carried out on recombinant repeat Tau which was purified by cation exchange chromatography. The ThS fluorescence analysis was used to study the aggregation kinetics of Tau. CD spectroscopy and electron microscopy were used to study the secondary structure and morphology of Tau respectively. The actin cytoskeleton modulation was studied in Neuro2a cells with help of immunofluorescent microscopy. Results showed that Toluidine Blue efficiently inhibited the formation of higher-order aggregates, which was evidenced by Thioflavin S fluorescence assay, SDS-PAGE, and TEM. Immunofluorescence studies on the cytoskeleton of Neuro2a cells showed that Toluidine Blue and photo-excited Toluidine Blue treatment at a non-toxic concentration of 0.5 µM stimulated the formation of actin-rich lamellipodia and filopodia structures. Tubulin networks were also differentially modulated after the treatment of Toluidine Blue and photo-excited Toluidine Blue. End-binding protein 1 (EB1) levels were observed to increase after Toluidine Blue and photo-excited Toluidine Blue treatment indicating accelerated microtubule polymerization. The overall study suggested that Toluidine Blue inhibited the aggregation of soluble Tau and photo-excited Toluidine Blue disaggregated the pre-formed Tau filaments. In our study, TB and PE-TB were observed to be potent against Tau aggregation. We observed a distinctive modulation of actin, tubulin networks, and EB1 levels after TB and PE-TB treatment, which suggested that TB and PE-TB have potency against cytoskeleton deformities.