Assay Development for Tau-directed Therapeutics

Neurofibrillary tangles (NFTs) are intracellular aggregates comprised of the microtubule-associated protein tau. These insoluble aggregates are found in the brain of those affected by Alzheimer’s disease and other related tauopathies. Advancements in cryo-electron microscopy over the last few years have revealed that structures of these aggregates found in various diseases are distinct from one another. They differ in terms of the amino acids participating in the folds of the aggregates, as well as the number and positioning of the beta-sheets. In vitro induced tau filaments commonly used to screen therapeutics have also been observed to have structures which are not similar to those found in diseases. We hypothesize that screening therapeutics against tau aggregates with structures similar to those observed in disease may allow for the identification of more effective therapeutics. In order to identify conditions to induce disease-relevant aggregates and develop assays to screen therapeutics, this study explores mechanical shaking-induced aggregation and RNA-induced aggregation, as well as the effect of a commonly observed post-translational modification in NFTs, hyperphosphorylation, on tau aggregation. We observed that mechanical shaking of various constructs of tau, which have been published to form AD-relevant structures, results in highly variable filament formation. These conditions also require extremely high concentrations of protein, which are not suitable for robust and reproducible assays to identify inhibition or disassembly of filaments. RNA-induced aggregation was investigated to induce aggregation in a reliable and reproducible manner at lower concentrations of protein. Although the formation of filaments with twists, an important characteristic of those found in disease, was observed with RNA-induced tau aggregation, extremely low filament formation with variability hinders the potential use of these conditions for tau-directed assay development. These results emphasize the need for the development of reliable assays at concentrations of protein suitable for the detection of changes in filament formation. Variants of pseudophosphorylated tau, used to mimic phosphorylation, which have been observed to form AD-relevant aggregates in vitro by others at high concentrations with shaking, generate reproducible filaments with observable twists with low-resolution microscopy, with polyphosphate-induced aggregation. Full-length pseudophosphorylated tau variants showed that the number of modified sites affects tau aggregation and microtubule stability. Incorporation of post-translational modifications in in vitro experiments may help better mimic and understand disease conditions, as well as help in identifying conditions and designing protocols for the generation of disease-relevant aggregates. These efforts may lead to a better understanding of these diseases and simultaneously lead to the identification of therapeutics for tau aggregation and help in finding a much-needed cure.

Author dc:creator

• Prasad, Veena

Contributors dc:contributor

• Gamblin, Truman
• Carless, Melanie
• Renthal, Robert
• Hsieh, Jenny
• Hopp, Sarah

• Aggregation
• Assay development
• MAPT
• Protein
• Tau

Handle dc:identifier.uri

https://hdl.handle.net/20.500.12588/7474

OAI identifier oai:identifier

oai:tdl-ir.tdl.org:20.500.12588/7474