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. 2020 Jul 23;10(1):12380.
doi: 10.1038/s41598-020-69403-2.

Photodynamic exposure of Rose-Bengal inhibits Tau aggregation and modulates cytoskeletal network in neuronal cells

Tushar Dubey  1   2 , Nalini Vijay Gorantla  1   2 , Kagepura Thammaiah Chandrashekara  3 , Subashchandrabose Chinnathambi  4   5
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V体育官网入口 - Photodynamic exposure of Rose-Bengal inhibits Tau aggregation and modulates cytoskeletal network in neuronal cells

Tushar Dubey et al. Sci Rep. .

Erratum in

Abstract

The intracellular Tau aggregates are known to be associated with Alzheimer's disease. The inhibition of Tau aggregation is an important strategy for screening of therapeutic molecules in Alzheimer's disease. Several classes of dyes possess a unique property of photo-excitation, which is applied as a therapeutic measure against numerous neurological dysfunctions. Rose Bengal is a Xanthene dye, which has been widely used as a photosensitizer in photodynamic therapy. The aim of this work was to study the protective role of Rose Bengal against Tau aggregation and cytoskeleton modulations. The aggregation inhibition and disaggregation potency of Rose Bengal and photo-excited Rose Bengal were observed by in-vitro fluorescence, circular dichroism, and electron microscopy. Rose Bengal and photo-excited Rose Bengal induce minimal cytotoxicity in neuronal cells VSports手机版. In our studies, we observed that Rose Bengal and photo-excited Rose Bengal modulate the cytoskeleton network of actin and tubulin. The immunofluorescence studies showed the increased filopodia structures after photo-excited Rose Bengal treatment. Furthermore, Rose Bengal treatment increases the connections between the cells. Rose Bengal and photo-excited Rose Bengal treatment-induced actin-rich podosome-like structures associated with cell membranes. The in-vivo studies on UAS E-14 Tau mutant Drosophila suggested that exposure to Rose Bengal and photo-excited Rose Bengal efficiency rescues the behavioural and memory deficit in flies. Thus, the overall results suggest that Rose Bengal could have a therapeutic potency against Tau aggregation. .

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Conflict of interest statement

The authors declare no competing interests.

V体育安卓版 - Figures

Figure 1
Figure 1
RB inhibits the Tau aggregation in vitro. (A) The domain organization of Tau. Tau is a natively unfolded protein having two domains, projection domain and a microtubule-binding domain. Rose Bengal is an anionic Xanthene dye widely used in clinical diagnosis. (B) The aggregation kinetics demonstrating a reduction in ThS fluorescence in samples incubated with various concentrations of RB. 40 µM RB showed maximum inhibition of Tau aggregation. (C) The graph showing the percentage of aggregation inhibition at the end of 72 h. (D) CD spectroscopy of RB treated samples, 40 µM RB and 20 µM RB observed to induce conformational changes in Tau aggregates. (E) The electron microscopy images of the RB treated sample have small broken fragments of Tau while the untreated sample has long tangled filaments.
Figure 2
Figure 2
PE-RB disaggregated the mature Tau filaments. (A) The schematic diagram demonstrating the irradiation assembly. A dark chamber was designed for irradiating the RB treated Tau aggregates where the green LED was used as a light source. (B) The SDS–PAGE analysis of Tau aggregates treated with various concentrations of RB. PE-RB was found to dissolve the aggregates as no higher-order bands were visible in the treated sample compared to untreated control (CC). The gel has been cropped for better representation of results. Raw SDS–PAGE is available with supplementary information. (C) The bar graph showing the decrease in ThS fluorescence in PE-RB treated samples indicating an efficient disaggregation of Tau filaments. (D) CD spectroscopic analysis of PE-RB treated Tau aggregates showed a shift in random coil region whereas the untreated aggregates have a dip near β-sheet structure. (E) The electron microscopy images of PE-RB treated samples showed broken Tau filaments; unlikely, the untreated samples have intact long filaments. p < 0.05, **p < 0.001, ***p < 0.0001 represents the statistical difference between control and treated groups.
Figure 3
Figure 3
The effect of RB on cell viability. (A) The MTT assay performed for observing the effect of various concentrations of RB on cell viability. RB showed minimal cytotoxicity up to 500 irradiation. (B) The PE-RB showed negligible cytotoxicity at a concentration of 500 nM. Whereas, the cells treated with 2.5 µM Tau aggregates also showed very minimal reduction in cell viability. p < 0.05, **p < 0.001, ***p < 0.0001 represents the statistical difference between control and treated groups.
Figure 4
Figure 4
Modulation of the cytoskeleton by RB. (A) The effect of RB and PE-RB was monitored on the cytoskeleton network in neuro2a cells. (B) The RB and PE-RB treated cells had extended axonal outgrowth with high tubulin intensity. (C) The quantification of neurite extension suggested that RB and PE-RB could modulate the tubulin cytoskeleton.
Figure 5
Figure 5
Modulation of the actin cytoskeleton by RB. (A) The effect of RB and PE-RB treatment on the actin cytoskeleton was monitored by immunofluorescence. (B) The immunofluorescence studies suggested that RB and PE-RB treatment generates various changes in actin cytoskeleton. (C) The quantification of images depicting the percentage of cells bearing lamellipodia in each experiment group. (D) The cells were quantified for the presence of filopodia using Image J software. The increased number of filopodia were observed in PE-RB treated cells.
Figure 6
Figure 6
RB and PE-RB increase podosome-like structure in cells. (A) The immunofluorescence studies for actin cytoskeleton modulation in presence of RB and PE-RB. The podosome-like structure in the cell membrane were observed after RB and PE-RB treatment. (B) The quantification of membrane-associated actin suggesting the increased localization of actin in the membrane after RB and PE-RB treatment.
Figure 7
Figure 7
Time of irradiation influences the cytoskeleton. (A,B) The cells were irradiated for different time points (10, 30 and 60 min). The 10 min and 30 min experimental set showed increased number of filopodia in treated cells. Cells subjected to an irradiation for 60 min showed decreased number of filopodia.
Figure 8
Figure 8
RB and PE-RB rescue the Memory and locomotor deficits. (A) UAS E-14 Drosophila flies treated with RB and PE-RB, various locomotor and olfactory assay was carried for observing the potency of RB against Tau toxicity. (B) A rescue in locomotor function after RB and PE-RB treatment was observed in negative geotaxis assay. (C) The larval crawling assay suggesting the positive regulation of locomotor activity by RB and PE-RB treatment. (D) Olfactory learning assay suggested that the cognitive function in Tau mutant flies were restored after RB and PE-RB treatment. (*p < 0.05, **p < 0.001, ***p < 0.0001, assigned for the difference between control and treated groups before photo-excitation. #p < 0.05, ##p < 0.001, ###p < 0.0001 represents the statistical difference between control and treated groups after photo-excitation.).
Figure 9
Figure 9
Rose Bengal attenuates the Tau toxicity. The RB attenuates Tau aggregation and photo-excited RB dissolves the Tau fibrils. The RB and PE-RB positively modulate the cytoskeleton. Memory and locomotor dysfunctions of UAS E14 Tau mutants were rescued after the RB and PE-RB treatment. Thus, RB was found effective against the various aspect of Tauopathy supporting it to be a neuroprotective molecule.
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