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

Abstract BackgroundAlzheimer’s disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. Inhibition of protein aggregation by photo-excited dyes is emerging as a novel strategy for the treatment of certain diseases. Toluidine Blue is a basic phenothiazine dye having the potency of photo-excitation by irradiation with a red light at 630±20 nm. 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. ResultsResults showed that Toluidine Blue efficiently inhibited the formation of higher-order aggregates which was evidenced by Thioflavin S fluorescence assay, SDS-PAGE and electron microscopy. Moreover, the photo-excited Toluidine Blue led to disaggregation of the mature repeat Tau fibrils, which were irradiated in the dark chamber customized in our lab. Further, studies on the effect of Toluidine blue on cell viability in Neuro2a cells using MTT assay showed that Toluidine Blue was not toxic to neuronal cells at lower concentrations but at high concentrations (> 5 µM) both Toluidine Blue and photo-excited Toluidine Blue induced significant toxicity. Immunofluorescence studies on the cytoskeleton of Neuro2a cells show 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 the 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. ConclusionsIn 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 clearly suggested that TB and PE-TB have potency against the cytoskeleton deformities. Thus, from the present work, we are stating that TB could be a potent molecule against Tauopathy.