Asparagine residue 368 is involved in Alzheimer's disease tau strain–specific aggregation

Shotaro Shimonaka,Shin-Ei Matsumoto,Montasir Elahi,Koichi Ishiguro,Masato Hasegawa,Nobutaka Hattori,Yumiko Motoi

doi:10.1074/jbc.ra120.013271

Abstract

In tauopathies, tau forms pathogenic fibrils with distinct conformations (termed "tau strains") and acts as an aggregation "seed" templating the conversion of normal tau into isomorphic fibrils. Previous research showed that the aggregation core of tau fibril covers the C-terminal region (243-406 amino acids (aa)) and differs among the diseases. However, the mechanisms by which distinct fibrous structures are formed and inherited via templated aggregation are still unknown. Here, we sought to identify the key sequences of seed-dependent aggregation. To identify sequences for which deletion reduces tau aggregation, SH-SY5Y cells expressing a series of 10 partial deletion (Del 1-10, covering 244-400 aa) mutants of tau-CTF24 (243-441 aa) were treated with tau seeds prepared from a different tauopathy patient's brain (Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration) or recombinant tau, and then seed-dependent tau aggregation was assessed biochemically. We found that the Del 8 mutant lacking 353-368 aa showed significantly decreased aggregation in both cellular and in vitro models. Furthermore, to identify the minimum sequence responsible for tau aggregation, we systematically repeated cellular tau aggregation assays for the delineation of shorter deletion sites and revealed that Asn-368 mutation suppressed tau aggregation triggered by an AD tau seed, but not using other tauopathy seeds. Our study suggested that 353-368 aa is a novel aggregation-responsible sequence other than PHF6 and PHF6*, and within this sequence, the Asn-368 residue plays a role in strain-specific tau aggregation in different tauopathies.

Highlights

Tau is a natively highly soluble, unfolded protein [1, 2], and physiologically it participates in the assembly and stabilization of microtubules (MTs) [3]
To explore the sequences of the tau C-terminal region that is responsible for aggregation, we constructed a series of plasmids expressing deletion mutants of tau-CTF24 (243–441 aa) lacking 16-amino acid residues: Del 1 (D244–259 aa), Del 2 (D259– 274 aa), Del 3 (D275–290 aa), Del 4 (D290–305 aa), Del 5 (D306–321 aa), Del 6 (D322–337 aa), Del 7 (D338–353 aa), Del 8 (D353–368 aa), Del 9 (D369–384 aa), and Del 10 (D385–400 aa) (Fig. 1A)
SH-SY5Y cells were transfected with these tau-CTF24 mutants (Del 3 to Del 10) or WT constructs and treated with tau seeds, which were mixed with a transfection reagent (Multifectam) for introduction into the cells

Summary

Introduction

Tau is a natively highly soluble, unfolded protein [1, 2], and physiologically it participates in the assembly and stabilization of microtubules (MTs) [3]. To explore the sequences of the tau C-terminal region that is responsible for aggregation, we constructed a series of plasmids expressing deletion mutants of tau-CTF24 (243–441 aa) lacking 16-amino acid residues: Del 1 (D244–259 aa), Del 2 (D259– 274 aa), Del 3 (D275–290 aa), Del 4 (D290–305 aa), Del 5 (D306–321 aa), Del 6 (D322–337 aa), Del 7 (D338–353 aa), Del 8 (D353–368 aa), Del 9 (D369–384 aa), and Del 10 (D385–400 aa) (Fig. 1A).

Results

Conclusion