30C50 l of brain lysates were resuspended in 10 volumes (v/v) of A68 buffer containing 1% sarkosyl and sonicated using an ultrasonic homogenizer for 1 min (power 25.0%). 8 mutant lacking 353C368 aa showed significantly decreased aggregation in both cellular and 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 brought on by an AD tau seed, but not using other tauopathy seeds. Our study suggested that 353C368 aa is usually 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. aggregation assay using recombinant tau mutants or peptides. We found that this sequence also affected the morphology of recombinant tau fibrils, but the sequence itself had no aggregation properties. In addition, we further investigated the smallest sequences involved in tau aggregation within 353C368 aa. We found that the aggregation property can be attributed to a single amino acid, Asn-368, whose mutations affected tau aggregation differently, depending on the type of tau strains. Results Seed-dependent tau aggregation assay of SH-SY5Y cells expressing tau-CTF24 deletion mutants 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 (243C441 aa) lacking 16-amino Rabbit polyclonal to AREB6 acid H-1152 residues: Del 1 (244C259 aa), Del 2 (259C274 aa), Del 3 (275C290 aa), Del 4 (290C305 aa), Del 5 (306C321 aa), Del 6 (322C337 aa), Del 7 (338C353 aa), Del 8 (353C368 aa), Del 9 (369C384 aa), and Del 10 (385C400 aa) (Fig. 1WT + seed) is usually shown in the plot. Data are means S.D. (= 3). **, 0.01; ***, 0.001 by Student’s test WT + seed. around the (magnified areas are indicated by the = 3). **, 0.01; ***, 0.001 by Student’s test WT + seed. Collectively, Del 5 (harboring the aggregation-prone PHF6 sequence), Del 8, and Del 9 showed significant decreases in tau-CTF24 aggregation using both biochemical and histochemical analyses. In vitro aggregation of recombinant tau-CTF24 deletion mutants To further analyze the effect of deletion aggregation of recombinant tau-CTF24 deletion mutants. = 3). Data values at 96 h were analyzed statistically using Student’s test the value of WT at 96 h (***, 0.001). aggregation assay of Del 6 and Del 9. Previous studies have exhibited that structural differences exist between heparin-induced tau filaments and those from brain tissues and that different seeding properties were observed between tau filaments seeded by AD tau and those assembled by heparin (29,C31). Hence, we speculated that tauopathy seed-induced cellular aggregation and heparin-induced aggregation occurred in a different manner, so that inconsistent results were obtained, even using the same tau deletion mutants. Deletion effects of 306C321 H-1152 (Del 5) and H-1152 353C368 (Del 8) aa H-1152 around the aggregation of full-length tau We further tested the effects of 306C321 (Del 5) and 353C368 (Del 8) aa deletions around the aggregation of full-length tau (2N4R and 2N3R isoforms). Cellular and aggregation experiments revealed that this 353C368 aa deletion in 2N4R and 2N3R tau caused decreases in tau aggregation and formation of abnormal fibrous structures (Fig. 5). These data showed that deletion of 353C368 aa affects the aggregation of tau, regardless of the length (C-terminal region or full length) and the presence or absence of microtubule-binding repeat 2 (2N4R or 2N3R). The results in Figs. 2C4 are summarized in Fig. 6and and and = 3). ***, 0.001 WT + seed. and aggregation was assessed using a ThS assay. Data are means S.D. (= 3). Data values at 96 h were analyzed statistically using Student’s test WT at 96 h (***, 0.001). and in the (determined by the cryo-EM method (26, 29)) and (trypsin-resistant fragments (32)) shows synthetic peptides derived from the deletion sequences. = 3). and = 3). **, 0.01; ***, 0.001 by Student’s test WT + seed. aggregation of a series of recombinant tau-CTF24 mutants harboring single aa deletions (366, 367, and 368). Aggregation was assessed using a ThS assay. Data are means S.D. (= 3). Data values at 96 h were analyzed statistically using Student’s test WT.