Tau, a microtubule-associated protein, aids in the maintenance of the axonal transport tract's integrity by promoting tubulin self-assembly into microtubule polymer. Tauopathies are characterized by abnormal tau buildup in the brain. To study tauopathy, this study uses a small tau fragment and its mutants to better understand tau aggregation and then uses that insight to develop aggregation inhibitors. First, a 20-mer peptide sequence (tau298-317) was selected from the longest tau isomer (2N4R). To emulate the disease conditions, two more mutants were selected and all were synthesized with solid-phase Fmoc chemistry. The phospo-mutant incorporated phosphate moiety at 305Ser (pS 305S), while Pro in 301 was replaced with Leu in P301L mutant. The kinetic analysis of the fragment showed that the fibrillation of the fragments required an external force such as palmitic acid (PA) to induce fibrillation. Interestingly, in the presence of PA, P301L fragment showed higher ThT fluorescence than wild-type tau298-317, while the incorporation of the phosphate group in pS 305S, completely revoked the aggregation. AFM studies also confirmed the presence of fibrils in PA facilitated wild-type tau298-317 and P301L systems, while no fibril was observed with pS 305S. CD study showed that tau298-317 and P301L adopted beta-sheet structures, while pS 305S maintained a random coil structure. In pS 305S, the incorporation of a negatively charged phosphate group might have inhibited the fibrillatory electrostatic interaction of PA with the peptide. Then, the aggregation inhibition of PA mediated tau298-317 system by the addition of a monovalent salt (NaCl) further confirmed the role of electrostatic interaction. We also tested the influence of hydrophobic interaction using 8-Anilinonaphthalene-1-sulfonic acid (ANS) dye. It showed concentration-dependent modest fluorescence rise. Thus, we proposed that the fibrillation process was mediated by both electrostatic and hydrophobic interaction, while electrostatic interaction plays a major role. Overall, a drug molecule must have the ability to inhibit both electrostatic and hydrophilic interactions to be a successful tauopathic drug. Various polyphenolic compounds such as resveratrol, rosmarinic acid, naringenin, EGCG, myricetin, and baicalein were tested as probable candidates. While multiple candidates were able to suppress the PA-mediated tau aggregation process, myricetin was highly effective in disintegrating preformed fibrils.

Palmitic acid mediated aggregation of the tau fragment: An in vitro fibrillation model for drug screening in tauopathies

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