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Updated: Jun 23, 2026
In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening
Published on: November 20, 2018
Three-/four-repeat-dependent aggregation profile of tau microtubule-binding domain clarified by dynamic light
Etsuko Sugino1, Chisato Nishiura, Katsuhiko Minoura
1Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan.
Abstract:
The analysis of the self-assembly mechanism of the tau microtubule-binding domain (MBD) could provide the information needed to develop an effective method for the inhibition of the tau filament formation because of its core region that forms the filament. The MBD domain in the living body consists of similar three or four 31- to 32-residue repeats, namely 3RMBD (R134) and 4RMBD (R1234), respectively. The filament formation of the MBD has been mainly investigated by fluorescence spectroscopy utilizing the beta-sheet structure-binding signal sensor thioflavin. This method observes the aggregation indirectly, and provides no information on the time-dependent change in aggregation size or volume. Thus, to determine the structure necessary for initiating MBD self-association, the dynamic light scattering (DLS) method was applied to the analysis of the aggregations of 3RMBD, 4RMBD and their component single repeats and shown to be a powerful tool for directly analyzing filament formation. DLS analysis clearly showed that the building unit for initiating the aggregation is the intermolecular R3-R3 disulfide-bonded dimer for 3RMBD and the intramolecular R2-R3 disulfide-bonded monomer for 4RMBD, and their aggregation processes under physiological condition differ from each other, which has not been clearly revealed by the conventional fluorescence method. The repeat-number-dependent aggregation model of MBD, together with the function of each repeat, reported in this paper should help to devise a method of preventing tau PHF formation.
Insights
Understanding tau microtubule-binding domain (MBD) self-assembly is key to inhibiting filament formation. Dynamic Light Scattering reveals distinct aggregation pathways for 3RMBD and 4RMBD, identifying critical dimer and monomer units.
Area of Science:
- Neuroscience
- Biochemistry
- Structural Biology
Background:
- Tau protein self-assembly into filaments is central to neurodegenerative diseases.
- The microtubule-binding domain (MBD) is the core region responsible for tau filament formation.
- Existing methods like fluorescence spectroscopy indirectly assess aggregation without revealing dynamic changes.
Purpose of the Study:
- To elucidate the self-assembly mechanism of tau MBD.
- To identify the specific structural units initiating MBD aggregation.
- To differentiate the aggregation pathways of different MBD variants.
Main Methods:
- Dynamic Light Scattering (DLS) was employed to directly analyze the aggregation of 3RMBD, 4RMBD, and their component repeats.
- DLS allowed for the direct observation of time-dependent changes in aggregation size and volume.
- Comparison with conventional fluorescence spectroscopy was performed.
Main Results:
- DLS directly demonstrated that the aggregation initiating unit for 3RMBD is an intermolecular R3-R3 disulfide-bonded dimer.
- DLS identified the aggregation initiating unit for 4RMBD as an intramolecular R2-R3 disulfide-bonded monomer.
- Distinct aggregation processes under physiological conditions were revealed for 3RMBD and 4RMBD, differing from previous findings.
Conclusions:
- The study provides a repeat-number-dependent aggregation model for MBD.
- Understanding the role of each repeat in aggregation is crucial for therapeutic strategies.
- These findings offer insights for developing methods to prevent tau filament (PHF) formation.
