Two-step screening method to identify α-synuclein aggregation inhibitors for Parkinson’s disease

Parkinson’s disease is a neurodegenerative disease characterized by the formation of neuronal inclusions of α-synuclein in patient brains. As the disease progresses, toxic α-synuclein aggregates transmit throughout the nervous system. No effective disease-modifying therapy has been established, and preventing α-synuclein aggregation is thought to be one of the most promising approaches to ameliorate the disease. In this study, we performed a two-step screening using the thioflavin T assay and a cell-based assay to identify α-synuclein aggregation inhibitors. The first screening, thioflavin T assay, allowed the identification of 30 molecules, among a total of 1262 FDA-approved small compounds, which showed inhibitory effects on α-synuclein fibrilization. In the second screening, a cell-based aggregation assay, seven out of these 30 candidates were found to prevent α-synuclein aggregation without causing substantial toxicity. Of the seven final candidates, tannic acid was the most promising compound. The robustness of our screening method was validated by a primary neuronal cell model and a Caenorhabditis elegans model, which demonstrated the effect of tannic acid against α-synuclein aggregation. In conclusion, our two-step screening system is a powerful method for the identification of α-synuclein aggregation inhibitors, and tannic acid is a promising candidate as a disease-modifying drug for Parkinson’s disease.

Cannabidiol Inhibits Tau Aggregation In Vitro

A hallmark of Alzheimer’s disease (AD) is the accumulation of tau protein in the brain. Compelling evidence indicates that the presence of tau aggregates causes irreversible neuronal destruction, eventually leading to synaptic loss. So far, the inhibition of tau aggregation has been recognized as one of the most effective therapeutic strategies. Cannabidiol (CBD), a major component found in Cannabis sativa L., has antioxidant activities as well as numerous neuroprotective features. Therefore, we hypothesize that CBD may serve as a potent substance to hamper tau aggregation in AD. In this study, we aim to investigate the CBD effect on the aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods in vitro and in silico. Using Thioflavin T (ThT) assay, circular dichroism (CD), atomic force microscopy (AFM), we demonstrated that CBD can suppress tau fibrils formation. Moreover, by quenching assay, docking and job’s plot, we further demonstrate that one molecule of CBD interacts with one molecule of tau protein through a spontaneous binding. Experiments performed by quenching assay, docking and Thioflavin T assay further established that the main forces are hydrogenvan der Waals and some non-negligible hydrophobic forces, affecting the lag phase of tau protein kinetics. Taken together, this study provides new insights about a natural substance, CBD, for tau therapy which may offer new hope for the treatment of AD.

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.

Two coexisting pseudo-mirror heteromolecular telomeric G-quadruplexes in opposite loop progressions differentially recognized by a low equivalent of Thioflavin T

The final 3′-terminal residue of the telomeric DNA G-overhang is inherently less precise. Here, we describe how alteration of the last 3′-terminal base affects the mutual recognition between two different G-rich oligomers of human telomeric DNA in the formation of heteromolecular G-quadruplexes (hetero-GQs). Associations between three- and single-repeat fragments of human telomeric DNA, target d(GGGTTAGGGTTAGGG) and probe d(TAGGGT), in Na+ solution yield two coexisting forms of (3 + 1) hybrid hetero-GQs: the kinetically favourable LLP-form (left loop progression) and the thermodynamically controlled RLP-form (right loop progression). However, only the adoption of a single LLP-form has been previously reported between the same probe d(TAGGGT) and a target variant d(GGGTTAGGGTTAGGGT) having one extra 3′-end thymine. Moreover, the flanking base alterations of short G-rich probe variants also significantly affect the loop progressions of hetero-GQs. Although seemingly two pseudo-mirror counter partners, the RLP-form exhibits a preference over the LLP-form to be recognized by a low equivalent of fluorescence dye thioflavin T (ThT). To a greater extent, ThT preferentially binds to RLP hetero-GQ than with the corresponding telomeric DNA duplex context or several other representative unimolecular GQs.