Probing the Influence of Single-Site Mutations in the Central Cross-β Region of Amyloid β (1–40) Peptides

Amyloid β (Aβ) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer’s disease. A complete mechanistic understanding how Aβ peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aβ40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aβ assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aβ40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aβ fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aβ fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aβ40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aβ40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aβ fibrillation pathway.

Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner.

The fluorescent benzothiazole Thioflavin T (ThT) has a high binding affinity to protein aggregates and is used as a marker for the study of this process, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilisation of ThT in research and diagnosis. Here, we show that ThT localises into the mitochondria of HeLa cells in a membrane-potential dependent manner. Specifically, ThT colocalised in cells with a well-established mitochondrial membrane-potential indicator Tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ=405 nm) exposure, but neither factor alone, caused depolarisation of mitochondrial membrane potential. This depolarisation effect was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its light-dependent binding in mitochondria. These results show that ThT can act as a membrane potential dye in mammalian cells, when used at low concentrations and with low blue-light exposure, while it causes dissipation of the mitochondrial membrane potential at higher concentrations and in the presence of blue light excitation. This conclusion motivates a re-evaluation of ThT use at micromolar range in live-cell analyses, while indicating that this dye can enable future studies on the potential connections between membrane potential dynamics and protein aggregation.

Thioflavin T fluorescence and NMR spectroscopy suggesting a non-G-quadruplex structure for a sodium binding aptamer embedded in DNAzymes

Recently, a Na+-binding aptamer was reported to be embedded in a few RNA-cleaving DNAzymes, including NaA43, Ce13d, and NaH1. The Na+ aptamer consists of multiple GG stretches, which is a prerequisite for the formation of G-quadruplex (G4) structures. These DNAzymes require Na+ for activity but show no activity in the presence of K+ or other metal ions. Given that DNA can selectively bind K+ by forming a G4 structure, this work aims to answer whether this Na+ aptamer also uses a G4 to bind Na+. Through comparative ThT fluorescence spectrometry studies, while a control G4 DNA exhibited notable fluorescence enhancement up to 5 mM K+ with a Kd of 0.28 ± 0.06 mM, the Ce13d DNAzyme fluorescence was negligibly perturbed with similar concentrations of K+. Opposed to this, Ce13d displayed specific remarkable fluorescence decrease with low millimolar concentrations of Na+. NMR experiments at two different pH values suggest that Ce13d adopts a significantly different conformation or equilibrium of conformations in the presence of Na+ versus K+ and has a more stable structure in the presence of Na+. Additionally, absence of characteristic G4 peaks in one-dimensional 1H NMR suggest that G4 is not responsible for the Na+ binding. This hypothesis is confirmed by the absence of characteristic peaks in the CD spectra of this sequence. Therefore, we concluded that the aptamer must be selective for Na+ and that it binds Na+ using a structural element that does not contain G4.

Autoxidation Enhances Anti-Amyloid Potential of Flavone Derivatives

The increasing prevalence of amyloid-related disorders, such as Alzheimer’s or Parkinson’s disease, raises the need for effective anti-amyloid drugs. It has been shown on numerous occasions that flavones, a group of naturally occurring anti-oxidants, can impact the aggregation process of several amyloidogenic proteins and peptides, including amyloid-beta. Due to flavone autoxidation at neutral pH, it is uncertain if the effective inhibitor is the initial molecule or a product of this reaction, as many anti-amyloid assays attempt to mimic physiological conditions. In this work, we examine the aggregation-inhibiting properties of flavones before and after they are oxidized. The oxidation of flavones was monitored by measuring the UV-vis absorbance spectrum change over time. The protein aggregation kinetics were followed by measuring the amyloidophilic dye thioflavin-T (ThT) fluorescence intensity change. Atomic force microscopy was employed to image the aggregates formed with the most prominent inhibitors. We demonstrate that flavones, which undergo autoxidation, have a far greater potency at inhibiting the aggregation of both the disease-related amyloid-beta, as well as a model amyloidogenic protein—insulin. Oxidized 6,2′,3′-trihydroxyflavone was the most potent inhibitor affecting both insulin (7-fold inhibition) and amyloid-beta (2-fold inhibition). We also show that this tendency to autoxidize is related to the positions of the flavone hydroxyl groups.

On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.

Comparison of Cerebrospinal Fluid Amyloidogenic Nanoplaques With Core Biomarkers of Alzheimer’s Disease

Accurate biomarkers of Alzheimer’s disease (AD) are essential for early diagnosis and intervention. Available biomarkers are not sufficient to permit the monitoring of AD progression over time, and additional biomarkers are required. Measures of aggregated amyloid-β (Aβ) could be useful biomarkers for AD. Here, we investigate whether levels of Thioflavin-T (ThT) positive amyloid aggregates, i.e., nanoplaques, in cerebrospinal fluid (CSF) could serve as useful biomarkers for AD. One-hundred and eighteen memory clinic patients were AT(N) classified, and CSF nanoplaque concentrations were compared between patients on the “Alzheimer’s continuum” (A+ patients) and patients with “Normal AD biomarkers” or “Non-AD pathologic change” (A− patients). CSF nanoplaque concentrations and sizes were quantified using the novel ThT-Fluorescence Correlation Spectroscopy (ThT-FCS) assay, and core biomarkers (Aβ42, total tau and phosphorylated tau) were determined by enzyme-linked immunosorbent assays. We investigated the association between nanoplaque concentrations and core biomarkers, and the diagnostic value of nanoplaque levels. Nanoplaque levels were increased in A+ patients compared to A− patients. Nanoplaque concentrations were negatively associated with Aβ42, but not related to total tau or phosphorylated tau measures. Quantification of nanoplaques did not improve the classification of patients on the Alzheimer’s continuum compared to the core biomarkers alone. Dynamic changes in nanoplaques concentration and size throughout AD stages should be explored in longitudinal studies.

Anti-amyloidogenic effect of artemin on α-synuclein

Abstractα-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson’s disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 μg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

Fusion Speed of Biomolecular Condensates

Biomolecular condensates formed through phase separation have a tendency to fuse. The speed with which fusion occurs is a direct indicator of condensate liquidity, which is key to both cellular functions and diseases. Using a dual-trap optical tweezers setup, we found the fusion speeds of four types of condensates to differ by two orders of magnitude. The order of fusion speed correlates with the fluorescence of Thioflavin T, which in turn reflects the macromolecular packing density inside condensates. Unstructured protein or polymer chains pack loosely and readily rearrange, leading to fast fusion. In contrast, structured protein domains pack more closely and have to break extensive contacts before rearrangement, corresponding to slower fusion. This molecular interpretation for disparate fusion speeds portends a unified understanding of the underlying physicochemical determinants.Entry for the Table of ContentsThe tendency of biomolecular condensates to fuse is key to cellular function and diseases. Using optical tweezers, fluorescence microscopy, and theoretical modeling, Ghosh and Zhou have begun to unravel the molecular origin for disparate fusion speeds among different biomolecular condensates. They found that fusion speed is dictated by macromolecular packing density inside condensates, which can be reported by ThT fluorescence.