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The absorption, fluorescence excitation and fluorescence spectra of ThT in aqueous solution and incorporated into amyloid fibrils. Panel a. Spectral characteristics of ThT in highly concentrated aqueous solution (ThT concentration was 28 mM). The absorption (red curve), fluorescence excitation (λ em = 490 and 570 nm, blue and red curves, respectively) and fluorescence (λ ex = 412 nm, red curve) spectra are presented. For comparison absorption spectrum of ThT in diluted aqueous solution (green curve) is given. Absorption spectrum, fluorescence excitation spectrum and fluorescence spectrum are given by solid, dashed and dotted lines, respectively. Panel b. Spectral characteristics of ThT bound to insulin amyloid fibrils. The absorption, fluorescence excitation (λ em = 490 nm) and fluorescence (λ ex = 450 nm) spectra are presented (magenta curves). Designations are the same as on the Panel A. Important, here is true absorption spectrum of ThT bound to amyloid fibrils obtained using solutions prepared by microdialyses after removal of free ThT absorption 49–52 , but not absorption spectrum of ThT in the presence of amyloid fibrils which is very close to absorption spectrum of free ThT molecules in aqueous solutions. For comparison the absorption, fluorescence excitation (λ em = 490 nm) and fluorescence (λ ex = 450 nm) spectra of free ThT in solution are presented (green curves).
Source publication
Fluorescence of thioflavin T (ThT) is a proven tool for amyloid fibrils study. The correct model of ThT binding to fibrils is crucial to clarify amyloid fibrils structure and mechanism of their formation. Although there are convincing evidences that ThT has molecular rotor nature, implying it’s binding to fibrils in monomer form, speculations conce...
Contexts in source publication
Context 1
... use Cary Eclipse spectrofluorimeter with horizontal slits and due to the developed in our laboratory method of correcting the fluorescence intensity 34 . Experimental data obtained for highly concentrated solutions of ThT filled the gap in our understanding of ThT spectral prop- erties. It was shown that fluorescence band with maximum at 570 nm (Fig. 8, Panel a) revealed for highly con- centrated ThT aqueous solutions can be attributed to ThT excimers. It was also shown that the ThT excimer fluorescence spectrum has nothing in common with the fluorescence of ThT incorporated into amyloid fibrils, which has maximа quite near that of ThT monomers in diluted solutions (Fig. 8, Panel b). ...
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... maximum at 570 nm (Fig. 8, Panel a) revealed for highly con- centrated ThT aqueous solutions can be attributed to ThT excimers. It was also shown that the ThT excimer fluorescence spectrum has nothing in common with the fluorescence of ThT incorporated into amyloid fibrils, which has maximа quite near that of ThT monomers in diluted solutions (Fig. 8, Panel b). Consequently, obtained data can be regarded as further confirmation of the idea that the increase in the ThT fluorescence intensity when the dye binds to amyloid fibrils can be explained only by the molecular rotor nature of ThT, and there is no basis to couple it with ThT excimers formation. Designations are the same as on the Panel ...
Citations
... Solutions were filtered consecutively through 220 nm and 50 nm syringe filters. Actual dye stock concentrations were determined from their optical absorption, using ε 412 = 31,600 M −1 cm −1 for ThT [40] and ε 590 = 87,000 M −1 cm −1 for CV [41], respectively. ...
Deposition of extracellular Amyloid Beta (Aβ) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer’s Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aβ and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aβ42 oligomers (AβOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.
... Most dyes for biomedical optical imaging (BOI) are charged molecules that are readily soluble in aqueous biological environments. One such example is the fluorescent dye Thioflavin T (ThT, Fig. 1A), which is able to detect amyloid β proteins (Aβ) in vitro, a distinguishing feature of certain neurodegenerative diseases such as Alzheimer's disease (AD) [17][18][19][20]. ThT shows higher fluorescence intensity in solutions containing Aβ fibrils compared to the control solutions without fibrils, which is due to steric immobilization of the dye at the Aβ binding site [21,22]. ...
... The obtained poses show a strong preference for binding within the hydrophobic cleft formed by Phe19, Ala21, Asn27, Ile31 hydrophobic sidechains, and backbones of Lys28-Ala30. The interior segment of the hydrophobic KLVFFA motif (amino acid sequence [16][17][18][19][20][21], which has often been proposed as a binding site on the amyloid, makes up one half of this cleft. Since the burial of the hydrophobic surface of the fluorophore within this cleft avoids the entropic penalty of ordering bulk water, this buried binding site is more probable than shallow binding on the amyloid fibril surface. ...
... It is reported that thioflavin-T (ThT) selectively binds to amyloid fibrils, enhancing their fluorescence intensity [38]. Si NWs exhibits a dose-dependent inhibitory effect on (40.5, 81, 162, and 324 µg/mL) co-incubated with Aβ 1-42 monomers were 78%, 63%, 42%, and 32% of the fluorescence intensity of the Aβ 1-42 monomer group, respectively ( Figure 2b). ...
... It is reported that thioflavin-T (ThT) selectively binds to amyloid fibrils, enhancing their fluorescence intensity [38]. Si NWs exhibits a dose-dependent inhibitory effect on Aβ1-42 fibrillization in vitro (Figure 2a). ...
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid β (Aβ) plaques in the brain. Aβ1-42, the major component of Aβ plaques, was toxic to neuronal cells. Si NWs have potential applications in preventing Aβ aggregation due to their small particle size, high specific surface area, and excellent biocompatibility. In this study, we investigated the effects of silicon nanowires (Si NWs) on the inhibition of Aβ1-42 fibril formation. Si NWs significantly reduced Aβ1-42 oligomer-induced cell death in PC12 cells, which was confirmed by MTT assay and FDA/PI double staining. Most importantly, pre-incubation of Si NWs largely prevents Aβ1-42-induced cell death in PC12 cells, suggesting that Si NWs produces anti-Aβ neuroprotection via the inhibition of Aβ aggregation. Our findings suggest that Si NWs could be a potential therapeutic agent for AD by protecting neuronal cells from Aβ1-42 toxicity.
... In Fig. 4, a comprehensive quantitative analysis is presented, utilizing the decomposition of the phasor plot data with a focus on two principal lifetime components. It is crucial to note that a similar analysis has previously been conducted in various systems, as referenced in the literature (31,51,75). This analytical approach represents the simplest model employed to elucidate the ThT fluorescence decay under the specified conditions. ...
The investigation of amyloid fibril formation is paramount for advancing our understanding of neurodegenerative diseases and for exploring potential correlated therapeutic strategies. Moreover, the self-assembling properties of amyloid fibrils show promise for the development of advanced protein-based biomaterials. Among the methods employed to monitor protein aggregation processes, fluorescence has emerged as a powerful tool. Its exceptional sensitivity enables the detection of early-stage aggregation events that are otherwise challenging to observe. This research underscores the pivotal role of fluorescence analysis, particularly in investigating the aggregation processes of hen egg white lysozyme, a model protein extensively studied for insights into amyloid fibril formation. By combining classical spectroscopies with fluorescence microscopy and by exploiting the fluorescence properties (intensity and lifetime) of the thioflavin T, we were able to noninvasively monitor key and complex molecular aspects of the process. Intriguingly, the fluorescence lifetime imaging-phasor analysis of thioflavin T fluorescence lifetime on structures at different stages of aggregation allowed to decipher the complex fluorescence decay behavior, highlighting that their changes rise from the combination of specific binding to amyloid typical cross-β structures and of the rigidity of the molecular environment.
... Several studies have reported the interaction of ThT with β-sheet structures and increased fluorescence intensity. However, it should be noted that ThT also induces fluorescence upon binding to some non-β-sheet rich structures, e.g. in transthyretin and acetylcholinesterase [45][46][47]. ...
The biological function of a protein is characterised by its three-dimensional conformation and encoded by its amino acid sequence. Tremendous effort has been devoted to understanding the mechanism of protein folding and how the amino acid sequences encode the correct functional conformation of a protein. Fluorescence-based methods, such as time-resolved spectroscopy, fluorescence correlation spectroscopy, or labelling of the proteins by external fluorescent dyes, have been employed to understand the protein folding dynamics. Herein, we describe recent fluorescence spectroscopy-based techniques used to study the conformational dynamics of a protein. Furthermore, these techniques are commonly available in most research laboratories and are used to study the protein–protein, protein–DNA, and protein–ligand interactions. We focus on the extrinsic fluorescent dyes to characterise the folding intermediates and detect the amyloid fibril aggregation in Alzheimer’s and Parkinson’s diseases.KeywordsFluorescence spectroscopyProtein foldingExtrinsic fluorescent dyesProtein aggregatesIntrinsic fluorophoreFluorescence anisotropy
... To calculate the ThT concentration, the molar extinction coefficient of 31600 1/(M·cm) at 410 nm was used [31]. The temperature in the experiments was controlled and set to 25 o C. ThT concentration was CThT = 70 μM in all the experiments. ...
... ThT fluorescence is widely used for histological staining of protein aggregates. 27,28 ThT emission has been shown to increase in proportion to Aβ concentration under a number of different experimental conditions, 29,30 including in live spheroids. 31 We found that ThT emission was significantly elevated in AD patient-derived spheroids and control spheroids treated with aggregated Aβ. ...
Cell culture systems derived from the progenitor cells of human patients have many advantages over animal models for therapeutic drug testing and studies of disease pathogenesis. Here we describe a three‐dimensional (3D) spheroid co‐culture system of neurons and astrocytes derived from induced pluripotent stem cells–neural precursor cells (iPSCs–NPCs) of Alzheimer's disease (AD) patients or healthy individuals that can provide information on drug efficacy unobtainable by 2D co‐culture or monoculture approaches. iPSCs–NPCs of healthy controls or AD patients were seeded onto 96‐well U‐bottom plates and incubated with neuronal differentiation medium for one week and with astrocytic medium for two weeks to replicate the temporal order of cell maturation during brain development. These 3D spheroid models expressed marker proteins for mature neurons and astrocytes. In particular, patient‐derived spheroids showed beta‐amyloid (Aβ) accumulation as revealed by thioflavin T (ThT) staining and ELISA. Aggregation of Aβ induced caspase activation and cell death, while the neuroprotectants nordihydroguaiaretic acid (NDGA) and curcumin (CU) reduced the levels of both ThT and caspase staining. Taken together, these results demonstrate the feasibility of our 3D spheroids combined with ThT and caspase staining as a patient‐based anti‐AD drug screening platform. (1) Three‐dimensional (3D) spheroids derived from iPSCs–NPCs are promising patient‐based models for efficient drug screening. (2) The 3D spheroids demonstrate remarkably consistent sizes and cellular compositions, indicating their suitability for reliable drug screening. (3) Spheroids derived from AD patient iPSCs–NPCs exhibit stunted neuronal dendrites, Aβ deposits, and reactive astrocytes similar to those in the AD brain, reflecting the disease conditions of patients. (4) ThT and caspase 3 fluorescence staining of spheroids allowed facile detection of changes in Aβ deposition and neuronal cell apoptosis.
... ThT fluorescence measurements of protein in the presence CTAB and CTAB-heparin mixture were performed by exploiting the Cary Eclipse fluorescence Spectrophotometer. Molar extinction coefficient of ThT dye is 31600 M −1 cm −1 at 412 nm which was applied to determine its concentration [35] . The measurements were executed in the 25 mM phosphate buffer at 7.0. ...
Heparin, a member of the glycosaminoglycan (GAG) family, is an extremely acidic sulfur-containing polysaccharide which has been suggested to be coupled with protein aggregation-related diseases including Alzheimer's, Parkinson's and Prion diseases. Here, we study the effect of heparin on the cetyltrimethylammonium bromide (CTAB)-induced aggregation of bovine serum albumin (BSA) exploiting biophysical approaches. Kinetic measurements indicated that heparin caused complete inhibition of CTAB-induced aggregation of protein with a significant decrease in the apparent rate constant. Far- and near-UV CD measurements depicted that CTAB behaves as a protein-aggregating agent. However, the perturbation of secondary and tertiary structure-induced by CTAB and the CTAB-induced amyloid formation of protein were prevented by heparin. ITC measurements revealed that enhancement of aggregation induced by CTAB was as a result of sequential binding in which CTAB binds with the protein and accompanied by exothermic heat of interaction. ITC measurements also showed that CTAB and heparin form complex while the complex does not bind with protein. Thus, we hypothesize that CTAB-Heparin complex is engaged in the inhibition of protein aggregation. Therefore, the mechanism of action CTAB-heparin complex in inhibition of protein aggregation may be used as a basis of the development of therapeutic strategies of various neurodegenerative complications.
... Thioflavin-T (ThT) could produce enhanced fluorescence when binds to amyloid fibrils [24]. Phloroglucinol could concentration-dependently inhibit Aβ 1-42 fibrillization in vitro (Fig. 1A). ...
Amyloid proteins, such as β-amyloid (Aβ) and α-synuclein (α-syn), could form neurotoxic aggregates during the progression of neurodegenerative disorders. Phloroglucinol, a clinical-used drug for treating spasmodic pain, was predicted to cross the blood brain-barrier and possesses neuroprotective potential. In this study, we have found, for the first time, that phloroglucinol inhibited the formation of amyloid aggregates, and degraded pre-formed amyloid aggregates with the similar efficacy as curcumin, a widely known amyloid aggregation inhibitor. Moreover, phloroglucinol decreased the seeding during aggregation process and inhibited the aggregation of Aβ1–42 with homocysteine (Hcy) seeds. Molecular docking analysis further demonstrated hydrophobic interactions and hydrogen bonds between phloroglucinol and Aβ1–42/α-syn. Furthermore, phloroglucinol inhibited amyloid aggregates-induced cytotoxicity in neuronal cells and prevented Aβ1–42 + Hcy aggregates-induced cognitive impairments in mice. All these results suggested that phloroglucinol possesses the ability to degrade pre-formed amyloid aggregates, to inhibit the seeding during amyloid aggregation, and to reduce the neurotoxicity, indicating the reposition possibility of phloroglucinol as a novel drug for treating neurodegenerative disorders.
... To monitor the aggregation of Aβ42 into Aβ fibrils, 10 µM Aβ42 peptides were added to phosphate-buffered saline (PBS, pH 7.4) at 37 • C and transferred onto 96-well plates (Bio-One, Greiner, Kremsmünster, Austria) containing 10 µM ThT. The fluorescence intensity at 485 nm was measured by an ELISA reader (Infinite 200Pro, Tecan, Männedorf, Switzerland) under an excitation wavelength of 440 nm, after 1, 4, 7, 11, and 14 days [59]. ...
Amyloid-β (Aβ) peptides play a key role in Alzheimer’s disease (AD), the most common type of dementia. In this study, a polysaccharide from Bletilla striata (BSP), with strong antioxidant and anti-inflammatory properties, was extracted using a low-temperature method and tested for its efficacy against AD, in vitro using N2a and BV-2 cells, and in vivo using an AD rat model. The characterization of the extracted BSP for its molecular structure and functional groups demonstrated the effectiveness of the modified method for retaining its bioactivity. In vitro, BSP reduced by 20% reactive oxygen species (ROS) levels in N2a cells (p = 0.0082) and the expression levels of inflammation-related genes by 3-fold TNF-α (p = 0.0048), 4-fold IL-6 (p = 0.0019), and 2.5-fold IL-10 (p = 0.0212) in BV-2 cells treated with Aβ fibrils. In vivo, BSP recovered learning memory, ameliorated morphological damage in the hippocampus and cortex, and reduced the expression of the β-secretase protein in AlCl3-induced AD rats. Collectively, these findings demonstrated the efficacy of BSP for preventing and alleviating the effects of AD.
