Fluorescent Thioflavin Research Articles

Docosahexaenoic acid disrupts in vitro amyloid β1‐40 fibrillation and concomitantly inhibits amyloid levels in cerebral cortex of Alzheimer’s disease model rats

We have previously reported that dietary docosahexaenoic acid (DHA) improves and/or protects against impairment of cognition ability in amyloid beta(1-40) (Abeta(1-40))-infused Alzheimer's disease (AD)-model rats. Here, after the administration of DHA to AD model rats for 12 weeks, the levels of Abeta(1-40), cholesterol and the composition of fatty acids were investigated in the Triton X100-insoluble membrane fractions of their cerebral cortex. The effects of DHA on the in vitro formation and kinetics of fibrillation of Abeta(1-40) were also investigated by thioflavin T fluorescence spectroscopy, transmission electron microscopy and fluorescence microscopy. Dietary DHA significantly decreased the levels of Abeta(1-40), cholesterol and saturated fatty acids in the detergent insoluble membrane fractions of AD rats. The formation of Abeta fibrils was also attenuated by their incubation with DHA, as demonstrated by the decreased intensity of thioflavin T-derived fluorescence and by electron micrography. DHA treatment also decreased the intensity of thioflavin fluorescence in preformed-fibril Abeta peptides, demonstrating the anti-amyloidogenic effects of DHA. We then investigated the effects of DHA on the levels of oligomeric amyloid that is generated during its in vitro transformation from monomers to fibrils, by an anti-oligomer-specific antibody and non-reducing Tris-Glycine gradient (4-20%) gel electrophoresis. DHA concentration-dependently reduced the levels of oligomeric amyloid species, suggesting that dietary DHA-induced suppression of in vivo Abeta(1-40) aggregation occurs through the inhibitory effect of DHA on oligomeric amyloid species.

Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter.

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.

Evidence for Stepwise Formation of Amyloid Fibrils by the Mouse Prion Protein

The full-length mouse prion protein, moPrP, is shown to form worm-like amyloid fibrils at pH 2 in the presence of 0.15 M NaCl, in a slow process that is accelerated at higher temperatures. Upon reduction in pH to 2, native moPrP transforms into a mixture of soluble β-rich oligomers and α-rich monomers, which exist in a slow, concentration-dependent equilibrium with each other. It is shown that only the β-rich oligomers and not the α-rich monomers, can form worm-like amyloid fibrils. The mechanism of formation of the worm-like amyloid fibrils from the β-rich oligomers has been studied with four different physical probes over a range of temperatures and over a range of protein concentrations. The observed rate of fibrillation is the same, whether measured by changes in ellipticity at 216 nm, in thioflavin fluorescence upon binding, or in the mean hydrodynamic radius. The observed rate is significantly slower when monitored by total scattering intensity, suggesting that lateral association of the worm-like fibrils occurs after they form. The activation energy for worm-like fibril formation was determined to be 129 kJ/mol. The observed rate of fibrillation increases with an increase in protein concentration, but saturates at protein concentrations above 50 μM. The dependence of the observed rate of fibrillation on protein concentration suggests that aggregate growth is rate-limiting at low protein concentration and that conformational change, which is independent of protein concentration, becomes rate-limiting at higher protein concentrations. Hence, fibril formation by moPrP occurs in at least two separate steps. Longer but fewer worm-like fibrils are seen to form at low protein concentration, and shorter but more worm-like fibrils are seen to form at higher protein concentrations. This observation suggests that the β-rich oligomers grow progressively in size to form critical higher order-oligomers from which the worm-like amyloid fibrils then form.

Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme–substrate complex and proceed to an acylated enzyme intermediate which is then hydrolyzed. However, the hydrolysis of the carbamoylated enzyme is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we show that the reaction of carbachol (carbamoylcholine) with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. These fluorescence changes allow not only the monitoring of the course of the carbamoylation reaction but also the determination of carbachol affinities for the A- and P-sites.

Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The 'amyloid cascade hypothesis' assigns the amyloid-beta-peptide (Abeta) a central role in the pathogenesis of AD. Although it is not yet established, whether the resulting Abeta aggregates are the causative agent or just a result of the disease progression, polymerization of Abeta has been identified as a major feature during AD pathogenesis. Inhibition of the Abeta polymer formation, thus, has emerged as a potential therapeutic approach. In this context, we identified peptides consisting of d-enantiomeric amino acid peptides (d-peptides) that bind to Abeta. D-peptides are known to be more protease resistant and less immunogenic than the respective L-enantiomers. Previously, we have shown that a 12mer D-peptide specifically binds to Abeta amyloid plaques in brain tissue sections from former AD patients. In vitro obtained binding affinities to synthetic Abeta revealed a K(d) value in the submicromolar range. The aim of the present study was to investigate the influence of this d-peptide to Abeta polymerization and toxicity. Using cell toxicity assays, thioflavin fluorescence, fluorescence correlation spectroscopy and electron microscopy, we found a significant effect of the d-peptide on both. Presence of D-peptides (dpep) reduces the average size of Abeta aggregates, but increases their number. In addition, Abeta cytotoxicity on PC12 cells is reduced in the presence of dpep.

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper-zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of approximately 650 kDa under physiological conditions, i.e., 37 degrees C, pH 7.0, and 100 microM protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.

A Designed Protein Interface That Blocks Fibril Formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures.

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein

In the brains of Alzheimer’s disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of β-structures or α-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris–HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the α-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10–30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an α-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of <10% or >40%, the intermediate conformation between the random and α-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native α-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.

Different associational and conformational behaviors between the second and third repeat fragments in the tau microtubule-binding domain.

The third repeat fragment (R3) in the four-repeat microtubule-binding domain of the water-soluble tau protein has been considered to play an essential role in the protein's filamentous assembly. To clarify the associational and conformational features that differentiate R3 from the second repeat, R2, the heparin-induced assembly profiles of these peptide fragments were monitored by the thioflavin fluorescence method and electron microscopy. The trifluoroethanol-induced reversible conformational change from a random structure to an alpha-helical structure, in an aqueous solution, was monitored by CD measurement, and the structure of R2 in trifluoroethanol solution was analyzed by a combination of two-dimensional 1H-NMR measurements and molecular modeling calculations to facilitate comparison with the structure of R3. The speed of R3 assembly was remarkably faster than that of R2, in spite of their similar amino acid sequences. The averaged NMR conformers of R2 exhibited the whole-spanning alpha-helical structure. Similar features observed in R2 and R3 conformers in trifluoroethanol were that the Leu10-Leu20/Lys20 sequence takes a helical structure with the amphipathic-like distribution of the respective side-chains, whereas the C-terminal moieties are both flexible. In contrast, a notable difference was observed at the N-terminal Val1-Lys6 sequence, namely, a helical conformation for R2 and an extended conformation for R3. These conformational behaviors would be associated with the different self-aggregation speeds and seeding reactions between R2 and R3.

Amyloid deposition in the hippocampus and entorhinal cortex: quantitative analysis of a transgenic mouse model.

Various transgenic mouse models of Alzheimer's disease (AD) have been developed that overexpress mutant forms of amyloid precursor protein in an effort to elucidate more fully the potential role of beta-amyloid (A beta) in the etiopathogenesis of the disease. The present study represents the first complete 3D reconstruction of A beta in the hippocampus and entorhinal cortex of PDAPP transgenic mice. A beta deposits were detected by immunostaining and thioflavin fluorescence, and quantified by using high-throughput digital image acquisition and analysis. Quantitative analysis of amyloid load in hippocampal subfields showed a dramatic increase between 12 and 15 months of age, with little or no earlier detectable deposition. Three-dimensional reconstruction in the oldest brains visualized previously unrecognized sheets of A beta coursing through the hippocampus and cerebral cortex. In contrast with previous hypotheses, compact plaques form before significant deposition of diffuse A beta, suggesting that different mechanisms are involved in the deposition of diffuse amyloid and the aggregation into plaques. The dentate gyrus was the hippocampal subfield with the greatest amyloid burden. Sublaminar distribution of A beta in the dentate gyrus correlated most closely with the termination of afferent projections from the lateral entorhinal cortex, mirroring the selective vulnerability of this circuit in human AD. This detailed temporal and spatial analysis of A beta and compact amyloid deposition suggests that specific corticocortical circuits express selective, but late, vulnerability to the pathognomonic markers of amyloid deposition, and can provide a basis for detecting prior vulnerability factors.

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.

Amyloid-like protein structure in mammalian ocular lenses

Over the past 30 years, extensive Raman and infrared spectroscopy studies determined that the major proteins in normal mammalian lenses exist predominantly as beta-pleated sheets in vivo. The present study examines the presence of amyloid protein supramolecular order of lens protein beta-pleated sheet arrays. The classic amyloidophilic stains Congo red and thioflavine were used in situ to identify lens regions with amyloid protein structure using brightfield microscopy. Birefringence in Congo red stained lenses was determined using polarizing light microscopy. Thioflavine stained lenses were also examined by fluorescence microscopy to detect a diagnostic fluorescence red-shift indicative of the intercalation of thioflavine into beta-sheet amyloid protein structures. The major findings are 1) Congo red staining begins in the lens interior at an abrupt boundary coinciding with the extensive reorganization of cell biology and physical environment that occurs during normal lens fiber cell differentiation. 2) Apple-green birefringence in Congo red stained lenses co-localizes with amyloidophilic dye affinity. 3) Thioflavine stains the lens interior beginning at the same boundary. 4) A red shift in thioflavine fluorescence was detected in the lens interior that co-localizes with amyloidophilic dye affinities and birefringence in organelle-free lens fibers. The present data demonstrating amyloidophilic dye binding, in situ red shift in thioflavine fluorescence, and apple-green birefringence provide evidence that lens protein beta-sheet arrays are organized in an amyloid-like supramolecular order in interior fiber cells of mammalian ocular lenses. The inherent stability of amyloid-like protein structure may contribute to the long-term structural integrity and transparency of the lens.

Amyloid-like protein structure in mammalian ocular lenses

Purpose. Over the past 30 years, extensive Raman and infrared spectroscopy studies determined that the major proteins in normal mammalian lenses exist predominantly as ß-pleated sheets in vivo. The present study examines the presence of amyloid protein supramolecular order of lens protein ß-pleated sheet arrays. Methods. The classic amyloidophilic stains Congo red and thioflavine were used in situ to identify lens regions with amyloid protein structure using brightfield microscopy. Birefringence in Congo red stained lenses was determined using polarizing light microscopy. Thioflavine stained lenses were also examined by fluorescence microscopy to detect a diagnostic fluorescence red-shift indicative of the intercalation of thioflavine into ß-sheet amyloid protein structures. Results. The major findings are 1) Congo red staining begins in the lens interior at an abrupt boundary coinciding with the extensive reorganization of cell biology and physical environment that occurs during normal lens fiber cell differentiation. 2) Apple-green birefringence in Congo red stained lenses co-localizes with amyloidophilic dye affinity. 3) Thioflavine stains the lens interior beginning at the same boundary. 4) A red shift in thioflavine fluorescence was detected in the lens interior that co-localizes with amyloidophilic dye affinities and birefringence in organelle-free lens fibers. Conclusions. The present data demonstrating amyloidophilic dye binding, in situ red shift in thioflavine fluorescence, and apple-green birefringence provide evidence that lens protein ß-sheet arrays are organized in an amyloid-like supramolecular order in interior fiber cells of mammalian ocular lenses. The inherent stability of amyloid-like protein structure may contribute to the long-term structural integrity and transparency of the lens.

Modification of β2-microglobulin with D-glucose or 3-deoxyglucosone inhibits Aβ2M amyloid fibril extension in vitro

β2-microglobulin (β2M) is a major constituent of amyloid fibrils (fAβ2M) deposited in patients with Aβ2M amyloidosis. Recently, advanced glycation end products (AGE) of β2M and fAβ2M have been suggested to play an important role in the pathogenesis of APβM amyloidosis. We first characterized the states of AGE modification of fAβ2M. Western blot analysis with a monoclonal anti-AGE antibody showed that purified fAβ2M was naturally modified with AGE. Immunohistochemical studies of amyloid-deposired tissue have revealed a patchy distribution of the AGE-modified area in the amyloid deposits. Then we modified β2-m either with D-glucose or with 3-deoxyglucosone (3-DG) and investigated the effect of these modification on fAβ2M extension in vitro, using the recently established first-order kinetic model of fAβ2M extension in vitro. Western blot analysis and enzyme linked immunosorbent assay with a monoclonal anti-AGE antibody showed that these sugar-modfied β2M contained AGE. During the incubation of fAβ2M with native β2-m at 37°C the fluorescence of thioflavin T increased without a lag phase and proceeded to equilibrium. On the contrary, only a slight increase in fluorescence was observed during the incubation of fAβ2M with sugar-modified β2M. Moreover, sugar-modified β2M exhibited a dose-dependent inhibitory effect on the extension reaction of fAβ2Mwith native β2M. These results may suggest that in some in vivo situations, the modification of β2-m with AGE could play an inhibitory role for the formation of fAβ2M.

B Cell Granule Peptides Affect Human Islet Amyloid Polypeptide (IAPP) Fibril Formationin Vitro

Islet amyloid polypeptide (IAPP) forms fibrils spontaneously. We examined whether other B cell granule peptides affect formation of β-pleated sheet fibrilsin vitrofrom human IAPP. Quantitative radioassay (radioactivity of soluble IAPP after adding125I-IAPP) and thioflavine fluorescence spectroscopy showed that insulin, C-peptide, and pancreastatin inhibited fibril formation by 60–100% at ratio 100:1 (peptide:IAPP), whereas at 1:10 or 1:100, i.e., IAPP in excess, a potentiated IAPP fibril formation was induced by the peptides. Semi-quantitative analysis by electron microscopy yielded similar effects. Thus, spontaneous IAPP fibrillisation is influenced by other B cell secretory granule peptides in a molar ratio dependent manner.