Asparagine residue 368 is involved in Alzheimer’s disease tau strain-specific aggregation

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 carboxy-terminal region (243–406) 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 tauopathy patient’s brain [Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD)] 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 Asn368 mutation suppressed tau aggregation triggered by an AD-tau seed, but not using other tauopathies seeds. Our study suggested that 353–368 aa is a novel aggregation-responsible sequence other than PHF6 and PHF6*, and within this sequence, the Asn368 residue plays a role in strain-specific tau aggregation in different tauopathies. in various regions structures


INTRODUCTION
Tau is a natively highly soluble, unfolded protein (1,2), and physiologically it participates in the assembly and stabilization of microtubules (MT) (3). In the adult human brain, tau has six isoforms from alternatively spliced products (0N4R, 0N3R, 1N4R, 1N3R, 2N4R, and 2N3R) which differ from one another in containing zero, one, or two Biochemically, AD filaments were composed of both 3R and 4R tau, whereas PSP and CBD showed only 4R tau. Experimental evidence showed that tau aggregates, prepared from AD, PSP, or CBD brains, or recombinant tau, can convert native tau into abnormal aggregated tau when internalized into cells, like prion protein (12)(13)(14). Therefore, the hypothesis that aggregated tau is propagated through a prion-like mechanism possibly explained the spread of neuropathologies and progression of neuronal death in not only AD but also in PSP and CBD (6,15). Furthermore, a recent study showed that tau aggregates may forms conformationally distinct structures in different tauopathy, which were termed as "tau strains", and different strains determine the seeding potency in cellular and animal tauopathy models (16,17).
Understanding of the mechanisms of tau aggregation is required in order to establish effective treatments for tauopathy. However, much about these mechanisms remains unclear. It by guest on November 4, 2020 http://www.jbc.org/

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Asn368 is responsible for AD-tau seeding 3 has been revealed that MBD was mainly responsible for the core-region of PHFs (18), and tau-C-terminal fragments are prone to aggregation (19)(20)(21). Our previous report showed that C-terminal tau fragments (tau-CTF24: 243-441) were deposited in human brains with tauopathies as well as in the brains of aged transgenic mice (Tg601) overexpressing wildtype human tau (22). In addition, genetic analysis of patients with tauopathies revealed that nearly Here, we attempted to identify aggregation-responsible sequences in the Cterminal region of tau using a cellular seeddependent aggregation model of tau-CTF24. To determine the key sequences of the tau Cterminal region systematically, we prepared 10 deletion mutants of tau-CTF24 that were expressed in SH-SY5Y cells and treated with tau aggregates prepared from a patient's brain or recombinant tau. Then, we identified Del 8 (353-368 aa) as a novel aggregation-responsible sequence, and this region overlapped with the core sequence of AD tau, which was recently determined by means of cryo-electron microscopy (cryo-EM) (26). The role of this sequence in tau aggregation was tested in an in vitro 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 353-368 aa. We found that the aggregation property can be attributed to a single amino acid, Asn368, whose mutations affected tau aggregation differently depending on the type of tau strains.  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)(30)(31). Hence, we speculated that tauopathy seed-induced cellular aggregation and heparininduced in vitro aggregation occurred in a different manner, so that inconsistent results were obtained, even using the same tau deletion mutants.

Seed
Deletion effects of 306-321 (Del 5) and 353-368   . We speculated that this discrepancy also reflected the distinct conformational differences between disease-derived tau and in vitro fibrils, as considered above. Taken together, we found that single amino-acid deletion of Gly366, Gly367, and Asn368 was sufficient to decrease tau-CTF24 aggregation in the cellular experiment using AD tau seed.
To exclude the contribution of deletion itself and assess the involvement of the sidechains of Gly366, Gly367, and Asn368 on tau aggregate formation, we constructed single amino-acid replacement mutants of tau-CTF24.
First, tau-CTF24 G366I and G367I were constructed in order to test the small side-chain effect of Gly by replacing it with an amino-acid by guest on November 4, 2020 http://www.jbc.org/

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Asn368 is responsible for AD-tau seeding 8 containing a bulky side-chain, such as Ile. Fig. 7E shows that the replacement of Gly366 and Gly367 by Ile did not affect tau aggregation, unlike Δ366 and Δ367. This suggested that in the Gly366 and Gly367 residues, the side-chain of Gly itself does not play an essential role in cellular tau aggregation induced by AD tau seed.
Next, to simplify the side-chain of Asn368, we constructed tau-CTF24 N368A, and to assess the specific roles of the Asn368 side-chain on tau aggregation, we also prepared tau-CTF24 N368D, N368Q, and N368L, in which the chemical structures of the side-chains resemble that of Asn To investigate whether AD brain lysate from another case of AD can reproduce our results, we first prepared new lysates from two other AD patients' brains (AD 2 and AD 3, Table   S2 and Fig. S1). The results with AD 2-and AD 3-treated cells were almost similar to the experiments in which AD 1 lysate was used as the tau seed (top 3 panels in Fig. 8A  Collectively, these data suggest that Gly366 and Gly367 play no essential roles in AD tau-induced cellular tau aggregation, but in the case of Asn368, the side-chain of this amino-acid is essential for the process.  (Table S2 and Fig. S1).
Surprisingly, a decrease in tau aggregation in the single amino-acid deletion mutants (Δ366, Δ367, and Δ368), which was observed in cells treated with AD seeds, was abolished in cells treated with confirmed that the deletion of Asn368 also affected the aggregation of full-length 2N4R tau induced by AD tau seeds, but not by other tauopathies or recombinant seeds (the results of FTDP-17 seeds were excepted because these seeds showed no aggregation property on 4R2N tau) (Fig. S4).
To confirm these results, we tested AD tau seed-specific effects on tau-CTF24 Δ368 using fluorescent immunocytochemical analysis. We These results indicated that Ser320, the amino-acid residue that is closely associated with Asn368 in the core structures of AD tau filaments, is also essential for tau aggregation induced by AD tau seed, but not by other tauopathies.

DISCUSSION
In this study, we identified 353-368 aa as a novel aggregation-responsible sequence of tau-CTF24 not be included in that specific region so that the deletion of these amino acids did not affect heparin-induced tau aggregation. We were not able to clearly explain the exact cause of these discrepancies at the molecular level, but our data seems to reflect the structural and biochemical differences between human-derived AD tau and heparin-induced in vitro filament, which have been discussed to date (30,31,46).In the cryo-EM structures of AD tau filament cores shown in (26) and Fig. 9A, 353-368 aa (Del 8) sequence ranges from the end of "β6" to the N-terminal tip of "β8", and this sequence is opposite to the region ranging from the middle of "β2" to "β4" strand (318-339 aa, which is almost correspond with Del 6: 322-337 aa)" in the core structure.
Focusing on the amino-acids Gly366, Gly367, and Asn368 of which deletion from tau-CTF24 caused decreased aggregation in cellular models; the two glycine residues are located in the spacer region between "β7" and "β8", and this region was at the sharp corner of the polypeptide chain.
We first speculated that glycine residues play an essential role in this narrow space by taking advantage of its minimal side-chain (one hydrogen atom), but contrary to our expectation, replacement of glycine residues with isoleucine, by guest on November 4, 2020 http://www.jbc.org/ Downloaded from a hydrophobic amino-acid that has a bulky sidechain, did not cause any disturbance of cellular tau aggregation induced by AD tau seed (Fig. 7E).
In the case of Asn368, this residue is located at the N-terminal tip of "β8" opposing "β2" sheet in the atomic model of the AD tau filament. Tau showed a smaller decrease in the aggregation effect than that with AD 1 (Fig. 8B), despite the fact that these AD tau seeds showed almost the same typical AD banding pattern (Fig. S1). This data might indicate that AD seeds from different patients share similar structural and biochemical properties (32,33), but they may not be completely identical, and in specific experimental conditions these subtle differences will manifest as different results. It is difficult to clearly determine the reason why this event occurred only in N368A mutants, but considering the fact that only N368A mutation shortened its sidechain among Asn368 substitution mutants, we speculated that substitution of Asn368 to Ala caused slightly increased local peptide flexibility around "Ala368" and this might affect the receptivity against different AD seeds. Taken together, these data indicated that the proper configuration of the amido group of Asn368 on tau aggregation processes was likely to be important for formation of tau fibrils.

By focusing on the structures around
Asn368, we observed that the side-chains of Val318 and Ser320 were arranged facing inwards toward Asn368 at the cross-β interface between "β1-2" and "β8" (only the Thr319 side-chain faced outwards) (Fig. 9A). As a result of Ala replacement, we showed that the S320A mutant significantly decreased AD seed-induced tau aggregation. In contrast, T319A showed no decrease and V318A, which seems to be the closest to Asn368, showed only a mild decrease in tau aggregation. These findings showed that Ser320 also participated in the tau aggregation and may be present at the interface between Asn368 and Ser320, such as hydrogen bonding between the amido group of Asn368 and hydroxyl-group of Ser320. However, we are unable to exclude the possibility that the S320A  (32,51) and showed different seeding potency in cellular and animal tauopathy models (16,17). These diversities are termed as "tau strains". In this study, tau-CTF24 mutants of Δ366, Δ367, Δ368, N368A/D/Q/L ( Fig. 8A and B) and S320A (Fig 9C) showed significantly decreased aggregation only when cells expressing these mutants were treated with AD tau strain, but not with recombinant 2N4R or other non-AD tauopathies strain. As for the difference between tau strains, recent cryo-EM studies clearly indicated that different tau strains have distinct core structures as "C-shaped" (AD) (26,33), "kinked-hairpin" (recombinant 4R2N tau) (29), "elongated structure" (Pick's disease) (52) and "four-layered fold" (CBD) (53,54). Considering these findings, we speculated that the side-chain of Asn368 (and possibly Ser320) is specifically involved in the formation of "C-shaped" core structure and thereby tau Asn368 mutant was not templated by Most previous studies of tau-based therapy used mutated tau constructs or in vitro heparin assay systems; however, we used human AD seeddependent cell models and referred to the result of cryo-EM analysis of human AD brain. In this respect, our data will be more accurate for searching for AD therapeutic targets. Collectively, we expected that antibodies, peptides, or other chemical compounds that specifically recognize and block 353-368 aa or Asn368 might be promising candidates as tau aggregation inhibitors.
In this study, we found that Asn368 of tau was an essential single amino-acid for the with a set of primers (Table S1).  (Table   S2).

Antibodies-The
Frozen brain tissues were cut into 0. Statistical analysis-All values in the figures are expressed as mean ± SD. Biochemical data were statistically analyzed using the unpaired, twotailed Student's t test. A p value ≤ 0.05 was considered to be statistically significant.

Data availability
All data are provided in manuscript and raw data are available upon request.  were detected with T46 antibodies, and relative intensity (vs WT + seed) is shown in the plot. Data are means ± SD (n = 3). **P < 0.01, ***P < 0.001 by Student's t-test versus "WT + seed". a.u., arbitrary unit, n.s., not significant.