Photo-excited Toluidine Blue disaggregates the Repeat Tau and modulates Cytoskeletal structure in neuronal cells

Abstract

Alzheimers 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 novel strategy for the treatment of certain diseases. Toluidine Blue is a basic phenothiazine dye having potency of photo-excitation by irradiation with red light at 630{+/-}20 nm. In present work, we studied the effect of Toluidine Blue and photo-excited TB on aggregation of repeat Tau in-vitro using Thioflavin S fluorescence assay, SDS-PAGE and electron microscopy. Results show that TB efficiently inhabited the formation of higher order aggregates. Moreover, the photo-excited TB led to disaggregation of the mature repeat Tau fibrils. Further, studies on the effect of Toluidine blue on cell viability and cytoskeleton network of Neuro2acells show that TB was not toxic to neuronal cells at lower concentrations but at high concentrations (> 5 M) both TB and photo-excited TB induced significant toxicity. Immunofluorescence studies on the cytoskeleton of Neuro2a cells show that Toluidine Blue and photo-excited Toluidine Blue treatment at non-toxic concentration of 0.5 M stimulated 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.