A unique tau conformation generated by an acetylation-mimic substitution modulates P301S-dependent tau pathology and hyperphosphorylation

Abnormal intracellular accumulation of aggregated tau is a hallmark feature of Alzheimer's disease and other tauopathies. Pathological tau can undergo a range of post-translational modifications (PTMs) that are implicated as triggers of disease pathology. Recent studies now indicate that tau acetylation, in particular, controls both microtubule binding and tau aggregation, thereby acting as a central regulator of tau's biochemical properties and providing avenues to exploit for potential therapies. Here, using cell-based assays and tau transgenic mice harboring an acetylation-mimic mutation at residue Lys-280 (K280Q), we evaluated whether this substitution modifies the neurodegenerative disease pathology associated with the aggregate-prone tau P301S variant. Strikingly, the addition of a K280Q-substituted variant altered P301S-mediated tau conformation and reduced tau hyperphosphorylation. We further evaluated neurodegeneration markers in K280Q acetylation-mimic mice and observed reduced neuroinflammation as well as restored levels of N-methyl-d-aspartate receptors and post-synaptic markers compared with the parental mice. Thus, substituting a single lysine residue in the context of a P301S disease-linked mutation produces a unique tau species that abrogates some of the cardinal features of tauopathy. The findings of our study indicate that a complex tau PTM code likely regulates tau pathogenesis, highlighting the potential utility of manipulating and detoxifying tau strains through site-specific tau-targeting approaches.

which tau pathology is implicated in neuronal loss and cognitive decline. Because tau is not mutated in the majority of tauopathy patients, abnormal tau post-translational modifications (or PTMs) are a plausible pathogenic mechanism underlying cognitive defects. Recently, we and others characterized tau acetylation as a PTM that regulates both normal tau function (1)(2)(3)(4) and tau aggregation (5)(6)(7). The sheer abundance of tau acetylation sites, which rivals the number of tau phosphorylation sites, combined with their striking preference for the lysine-rich microtubule-binding region (MTBR), supports acetylation as a critical PTM controlling many of tau's biochemical properties.
Analysis of mouse and human brains strengthened a role for tau acetylation in vivo (1,2,4,8). In normal WT mice, acetylation of endogenous mouse tau was detected (8), suggesting tau acetylation is not restricted only to the diseased state. However, in transgenic AD models, acetylated tau was elevated, including, for example, in a bigenic mouse model of combined A␤ plaque and tau pathology, resulting in significantly increased acetylated tau and reduced overall survival (1,4,9,10). Similarly, injection of pre-formed tau fibrils, acting as prion-like tau seeds, led to rapid induction of tau acetylation in the hippocampus of tau transgenic mice only 2 weeks after fibril injections (11,12). In the human brain, accumulation of acetylated tau has consistently been observed in tau lesions from AD patients as well as a range of other human tauopathies, including familial frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy (1,(13)(14)(15).
Although these correlative studies link acetylated tau to tauopathies, the implications of acetylation required additional in vivo studies. Indeed, analysis of acetylation-mimic mutations either in the proline-rich (K174Q) or MTBR (K274Q/K281Q) regions suggested reduced tau turnover, synaptic defects, neurodegeneration, and memory loss (6,7). In contrast, however, other tau acetylation sites were suggested to be protective, including at the KXGS motifs (e.g. Lys-259, Lys-290, Lys-321, and Lys-353) present in each of tau's four MTBR domains (2,16). KXGS acetylation (on the underlined lysine) was shown to hinder phosphorylation at MARK-2 target sites (KXGS) (on the underlined serine), the latter of which accumulates in AD brain. Therefore, acetylation at distinct lysines cro ARTICLE spanning the tau protein could yield different phenotypes by influencing tau conformation, tau binding to MTs, modulation of tau turnover, and/or PTM priming events that alter nearby tau modifications.
Over the last few years, we performed a detailed characterization of acetylated tau at residue Lys-280 in the 2nd MTBR domain. This site was of particular interest for the following reasons. First, using an acetylation-specific antibody, tau Lys-280 acetylation was prominent in all human 4R-tauopathies but was not detectable in any control nondiseased brains, indicating a robust disease-specific marker of tauopathy (1). Second, Lys-280 acetylation-mimics reduced tau binding to MTs (17), consistent with prior reports that residue Lys-280 directly engaged MTs (18) acting as a regulatable switch that controls MT binding. Third, Lys-280 acetylation-mimics significantly enhanced tau aggregation kinetics in vitro (17), an effect that was recently confirmed by site-specific chemical acetylation only at residue Lys-280 (19). Finally, we found that CBP-mediated acetylation of tau at multiple sites, including Lys-280, led to a reduction in tau phosphorylation (rather than tau hyperphosphorylation) at AD-relevant phospho-tau epitopes (17), leaving open the question of whether Lys-280 acetylation promotes tau pathology. In fact, some tau modifications are known to abrogate tau binding to MTs yet also suppress tau aggregation, suggesting PTM-induced loss of normal tau function and gain of toxic tau aggregation are not always directly correlated and could be separable processes (20,21).
Here, we performed cell-based assays and developed a new tau transgenic mouse model expressing a Lys-280 acetylation-mimic mutation (K280Q) to interrogate the impact of an acetylation mimetic on tau pathogenesis. Rather than enhancing tau pathology, our results indicate that, in the context of a familial MAPT mutation, a tau Lys-280 acetyla-tion-mimic alleviates some of the features of P301S-induced neurodegeneration. Overall, our study suggests that a comprehensive understanding of tau PTMs could be exploited for site-specific targeting approaches to potentially slow taumediated neurodegeneration.

Acetylation-mimic tau at residue Lys-280 promotes tau hypo-phosphorylation
Our recent study showed that global CBP-mediated tau acetylation results in reduced tau phosphorylation at several AD-associated epitopes (17). We therefore asked whether only a single Lys-280 acetylation event was responsible for this observation. We generated constructs expressing acetylation mimic (K280Q) or nonmimic (K280R) mutants and expressed them in QBI-293 cells followed by immunoblotting with a panel of tau antibodies (Fig. 1A). Surprisingly, the K280Q acetylationmimic showed an ϳ5-fold reduction in pSer-202/Thr-205 (AT8) immunoreactivity (Fig. 1B). We also tested phospho-tau antibodies against more C-terminal phospho-tau epitopes, including pSer-396 and pSer-404, which detected phospho-tau species with differing gel mobility ranging from ϳ60 to 70 kDa (Fig. S1). Although the overall pSer-396 or pSer-404 immunoreactivity was unaffected by the K280Q mutation, the most slowly migrating ϳ70-kDa tau bands were reduced (Fig. S1A). We confirmed that the up-shifted ϳ70-kDa tau band was indeed due to tau hyper-phosphorylation using -phosphatase assays (Fig. S1B). These data support a preferential effect of the Lys-280 acetylation-mimic on hyper-phosphorylated tau. Thus, either global tau acetylation mediated by acetyltransferase activity or an acetylation-mimic mutation targeting a single residue in the 2nd repeat (Lys-280) is capable of altering Figure 1. A tau acetylation mimic at Lys-280 promotes tau dephosphorylation at multiple epitopes. A, QBI-293 cells expressing wildtype (WT) tau (2N4R tau isoform), Lys-280 acetylation-mimic tau (K280Q), or Lys-280 acetylation nonmimic tau (K280R) were harvested, and immunoblot analysis was performed using the indicated tau antibodies to detect phosphorylated tau at residues Ser-202/Thr-205 (AT8), total tau (K9JA), and GAPDH as a loading control. B, quantification of immunoblots from A showing that phosphorylated tau is reduced by K280Q. Statistical significance was assessed using a Student's t test from n ϭ 3 biological replicates (*, p Ͻ 0.05; **, p Ͻ 0.01; ns ϭ not significant). Error bars represent Ϯ S.E.

A tau K280Q mutation suppresses P301S pathology
tau's phosphorylation profile. In light of these findings, we sought to evaluate the consequences of the K280Q mutation in vivo using a new tau transgenic mouse model.

Characterization of acetylation-mimic tau transgenic mice
The well-characterized tau PS19 transgenic line expresses full-length 1N4R human tau containing the P301S mutation driven from the mouse prion promoter (22). These mice show ϳ20 -50% survival by 12 months of age due to the accumulation of mature tau pathology and neurodegeneration that is prominent in the hippocampal and cortical regions. Given that K280Q, but not K280R, led to altered tau phosphorylation profiles, we used the PS19 targeting vector to generate new transgenic lines containing an acetylation mimic (K280Q) substitution ( Fig. 2A), which we refer to hereafter as PS-KQ mice. Three lines of PS-KQ mice on a C57BL/6 background were generated by standard pronuclear injections. We generated low-(line 6), middle-(line 12), and high (line 13)-expressing PS-KQ lines and will focus the analysis below on line 13 because the tau transgene levels in these mice were more comparable with parental PS5 or PS19 mice (22).
Cohorts of PS19 and PS-KQ mice were aged to evaluate neurodegenerative phenotypes and generate survival curves.
PS-KQ mice did not develop any apparent signs of neuronal loss, showed no overt cognitive phenotypes (including hindlimb clasping and kyphosis, which are common in PS19 mice), and lived a normal life span that was indistinguishable from WT littermates (Fig. 2B). This was despite the fact that the PS-KQ mutant tau transgene is well-expressed throughout the brain. The lack of an overt phenotype contrasted with agematched PS19 mice, which showed progressive neurodegeneration and ϳ50% survival by 12 months of age (Fig. 2B, red line). We did not observe neurodegenerative phenotypes in any PS-KQ transgenic mice expressing varying levels of the tau transgene (lines 6, 12, and 13), although line 13 was the major focus of this study.
Immunohistochemistry (IHC) analysis of PS-KQ mice using a human tau-specific antibody (tau12) showed prominent tau expression and distribution in all brain regions analyzed with comparable immunoreactivity and staining intensity compared with PS19 mice (Fig. 2C). We observed expression in the hippocampus, particularly in the dentate granule layer, in which acetylation-mimic tau accumulated within neuronal soma and the surrounding neuropil, similar to PS19 mice. Immunoblotting of the isolated hippocampus showed acetylation-mimic transgene expression in all replicate brains (Fig. 2D). Although , and PS-KQ (n ϭ 25) mice, and statistical significance was assessed using the log rank test. WT and PS-KQ mice show 100% survival at 12 months, and these plots overlapped on the survival curve. PS19 mice show ϳ50% survival by 12 months of age (****, p Ͻ 0.001), whereas PS-KQ mice do not show any accelerated mortality, with a life span similar to normal WT mice. C, hippocampal and cortical (top panel) regions from 12-month-old mice were analyzed by IHC using the human tau-specific tau12 antibody, which detects transgenic human tau but not endogenous mouse tau. Representative images were taken from 12-month-old WT, PS19, and PS-KQ mice (n ϭ 6 per genotype). Scale bars ϭ 100 m. Higher magnification images of the cortex (middle panel) and hippocampus (Hipp, bottom panel) reveal PS-KQ tau accumulation in the neuronal soma of dentate granule (DG) neurons. D, isolated hippocampi from 12-month-old mice were fractionated into soluble and insoluble fractions and immunoblotted with the tau12 antibody. E, total soluble brain lysates from hippocampus, cortex, and brainstem were similarly immunoblotted with the tau12 antibody. Representative images are shown from a total of n ϭ 12 mice analyzed per genotype.
faster migrating lower molecular mass tau bands were consistently detected in PS-KQ mice, the highest molecular mass (ϳ70 kDa) tau bands corresponding to hyper-phosphorylated 1N4R human tau were nearly absent (Fig. 2D, soluble fraction). Biochemical fractionation showed a reduction in insoluble tau corresponding to pathologically-aggregated tau (Fig. 2D, insoluble fraction). Similar PS-KQ tau banding patterns were observed in other brain regions, including the cortex and brain stem, which harbored predominantly faster migrating tau bands, reflecting hypo-phosphorylated tau (Fig. 2E). Reduced accumulation of tau fragments ranging from ϳ25 to 50 kDa was also observed in the PS-KQ brain (Fig. 2E, see cleaved fragments). We attribute these differences to altered tau processing. In contrast to IHC analysis, which showed similar staining intensity, immunoblotting of full-length soluble human tau showed a trend toward slightly lower PS-KQ expression levels. We note that insoluble tau was consistently reduced in PS-KQ brains. These findings suggest that altered tau PTM profiles and/or tau conformation may underlie the lack of pathology and neurodegeneration in PS-KQ mice.

tau hypo-phosphorylation in acetylation-mimic tau transgenic mice
We next evaluated whether the absence of slower-migrating tau bands in PS-KQ mice reflected tau dephosphorylation. Isolated cortex (Fig. 3A) and hippocampus (Fig. 3B) were analyzed by immunoblotting with phospho-tau antibodies. We note that the corresponding total tau expression levels are depicted in Fig. 2. Despite soluble tau levels being readily detectable, phosphorylated tau was nearly absent in PS-KQ mice at all epitopes examined, notably Ser-202/Thr-205 (AT8), which was eliminated, whereas Ser-396 -positive phosphorylated tau bands were slightly detectable in PS-KQ mice but significantly reduced (Fig. 3C). Similarly, IHC analysis showed a strong reduction in phospho-tau pathology at AT8 and AT180 epitopes, both in neuronal soma and processes, with near-complete loss of AT8 immunoreactivity in PS-KQ mice (Fig. 3D). IHC analysis of the entire hippocampus is depicted in Fig. S2.
Given the reduction in phospho-tau levels, we considered the possibility that PS-KQ tau undergoes conformational changes resulting in dephosphorylation and reduced tau aggregation.

Figure 3. PS-KQ acetylation-mimic tau is predominantly hypo-phosphorylated.
A and B, hippocampal (Hipp) and cortical brain homogenates from 12-month-old mice were analyzed by immunoblotting using the indicated phosphorylation-specific tau antibodies that detect mid-domain (p-S202/T205) or C-terminal (p-S396) phospho-tau epitopes, both of which were reduced in PS-KQ mice, as shown by densitometry analysis of cortical immunoblotting data in C. The corresponding total tau expression from identical triplicate samples are depicted in Fig. 1D. Statistical significance was assessed using a Student's t test from n ϭ 3 biological replicates (***, p Ͻ 0.005). Error bars represent Ϯ S.E. D, brain sections from 12-month-old mice were analyzed by IHC using phosphorylation-specific tau antibodies, further confirming the reduction in phosphorylated tau in PS-KQ mice. Images depicting the entire hippocampus are shown in Fig. S2. Scale bar ϭ 100 m.

A tau K280Q mutation suppresses P301S pathology
We evaluated pathological tau using the conformation-dependent MC1 antibody, which detects a discontinuous epitope mediated by N-and C-terminal tau interactions (23,24). We also assessed tau amyloid conformation using thioflavin-S (ThS) staining. As shown in Fig. 4A, PS19 mice produce widespread MC1-positive inclusions in hippocampal and cortical regions, as reported previously (12,25). However, MC1 immunoreactivity was completely absent in PS-KQ mice despite widespread human tau transgene expression. MC1 analysis of the entire hippocampus is depicted in Fig. S2. Because the K280Q mutation lies just outside the MC1 epitope (residues 312-322), the lack of MC1 immunoreactivity suggests a con- formational alteration rather than steric interference with the MC1 epitope. Consistent with this possibility, ThS-positive aggregated tau lesions were also not detected in any brain regions in PS-KQ mice, in contrast to the amyloid-positive structures observed in the soma of PS19 mice (Fig. 4B).
To further confirm that PS-KQ generates a conformational change, we evaluated a panel of 1N4R tau (tau-T34) constructs in transfected QBI-293 cells. We found that MC1 immunoreactivity was reduced in cells expressing the double PS-KQ mutant compared with the single mutant (P301S), consistent with a stable conversion of tau conformation even in transiently expressed models (Fig. 5, A and B). The MC1 conformation is thought to precede pathological caspase-cleaved tau at residue Asp-421 during AD progression (26,27). Consistent with this predicted scheme, PS-KQ-transfected cells also showed reduced cleaved tau, detected with the tauC3 antibody, by immunofluorescence (Fig. 5, C and D) and immunoblotting (Fig.  5, E and F). Indeed, cleaved tau is also detectable in degenerating PS19 mice but not PS-KQ mice (Fig. 5G). Thus, the K280Q mutation causes a stable conformational change that partly abrogates P301S-mediated tau pathogenesis.
To further evaluate the aggregation propensity of PS-KQ, we analyzed tau mutants using an independent tau-seeding assay in which the introduction of repeat-domain-containing fibrillar tau seeds is sufficient to promote the aggregation and hyperphosphorylation of ectopically expressed full-length tau. P301L tau seeds were packaged and delivered into cultured QBI-293 cells expressing WT 1N4R-tau or mutant tau (P301S, K280Q, or PS-KQ), and seeded tau pathology was assessed by immunoblotting of soluble (Fig. 5H, top panel) and insoluble (Fig. 5H, bottom panel) fractions. Full-length P301S tau was optimally seeded leading to robust AT8-positive tau aggregation in the insoluble fractions (Fig. 5H, lanes 4 -6). Although tau seeds led to PS-KQ tau phosphorylation in the soluble fraction, there was reduced conversion of full-length tau into the insoluble fraction compared with P301S tau (Fig. 5H, compare lanes 4 -6 to 10 -12). PS-KQ tau showed ϳ50% reduced seeding based on the quantification of insoluble tau (Fig. 5I). Thus, using both mouse models and cell-based assays, we conclude that a K280Q acetylation-mimic insertion, placed in the context of a known pathogenic P301S mutation, enhances susceptibility to tau dephosphorylation and abrogates pathological tau aggregation.

Neurodegeneration markers in acetylation-mimic tau transgenic mice
Because gliosis is prominent in AD as well as mouse models of tauopathy, including the PS19 line, we evaluated mouse brains with antibodies to glial fibrillary acidic protein (GFAP), used to mark reactive astrocytes. As shown in Fig. 6A, PS19 mice show a marked increase in GFAP staining in the white and gray matter of the hippocampus. The astrogliosis closely matched the distribution and density of human tau-expressing neurons in the hippocampus. PS-KQ mice showed reduced astrogliosis, which appeared more similar to WT mice (Fig. 6B). However, we did note a subtle increase in GFAP staining in PS-KQ versus WT mice, indicating low baseline astrogliosis in PS-KQ mice. Excessive microglial activity has also been implicated in AD, in part via synaptic pruning, neuronal loss, and neurodegeneration (28). We therefore evaluated microgliosis using Iba1 antibodies and observed infiltrating Iba1-positive microglia in degenerating PS19 hippocampus, with apparent increases in both Iba1 signal intensity and Iba1-positive cell counts (Fig. 6, C and D). We highlight examples of amoeboid rounded microglia present in the PS19 hippocampus (Fig. 6E), a morphology associated with inflammation and neuronal damage. In contrast, PS-KQ mice showed minimal amoeboid morphologies and instead were enriched with the elongated ramified morphology (Fig. 6, E and F), consistent with the lack of neurodegeneration in these mice.
Given the limited neuroinflammation, we next asked whether the PS-KQ mutant tau was associated with reduced excitoxicity, a deregulated process implicated in neuronal dysfunction in AD. Aberrant tau re-localizes from the axon to the somatodendritic compartment, alters synaptic architecture, activates N-methyl-D-aspartate receptors (NMDARs), and promotes excessive calcium accumulation leading to synapse loss and cognitive decline (29). Thus, we interrogated whether PS-KQ tau was linked to excitotoxicity, as in PS19 mice, by evaluating parameters of synaptic dysfunction. We analyzed tau re-localization by double-labeling immunofluorescence with the dendritic marker MAP2. As shown in Fig. 7A, human tau (detected with human-specific tau-Y9 antibody) co-localized with MAP2-positive dendrites in both PS19 and PS-KQ hippocampal sections. Similar to the immunoblotting results above, transgenic tau levels are quite comparable in both PS19 and PS-KQ mouse brains. In fact, we often observed more robust human tau immunoreactivity in the neuronal soma of PS-KQ mice, consistent with the observations made by IHC (Fig. 2). These results indicate that PS-KQ tau retains its ability to re-localize to the dendritic compartment but nevertheless remains nontoxic.
We next evaluated synaptic integrity in brain lysates from PS19 and PS-KQ mice. Excitotoxicity is mediated partly by overactive NMDARs leading to excessive entry of calcium that activates downstream-signaling pathways. Activated NR2B subunits were previously shown to undergo proteolytic cleavage under excitotoxic conditions, producing N-terminal (ϳ115 kDa) and C-terminal (ϳ65 kDa) NR2B fragments (30 -32), a process regulated by the scaffolding protein post-synaptic density-95 (PSD-95), whose levels dictate susceptibility of NR2B to cleavage (30,33). We examined NR2B and PSD-95 as markers of tau-mediated excitotoxicity. Using a phospho-NR2B antibody that selectively recognizes the active phosphorylated (Y1472) form, we detected a prominent cleaved ϳ65-kDa NR2B fragment in 12-month-old PS19 mice in both hippocampal and cortical lysates, but not in WT or PS-KQ mice (Fig. 7B,  upper panel). NR2B fragmentation was associated with reduced levels of the ϳ180-kDa full-length NR2B subunit, indicative of excitotoxic NR2B cleavage in PS19 mice. Consistent with NR2B-mediated synaptic degeneration, reduced PSD-95 levels were also observed in PS19 mice, but not PS-KQ mice (Fig. 7B,  lower panel). Quantification of these findings in PS19 mice showed a significant reduction in full-length NR2B and PSD-95 with a simultaneous increase in NR2B fragmentation; none of these alterations were observed in PS-KQ mice (Fig. 7C). Overall, these data support reduced excitotoxic signaling in PS-KQ A tau K280Q mutation suppresses P301S pathology

Figure 6. Reduced astrogliosis and microgliosis in PS-KQ acetylation-mimic mice.
A, hippocampal CA1 images are shown depicting GFAP-positive astrocytes (green). An increase in astrocyte activity is detected in degenerating the hippocampus in 12-month-old PS19 mice, which was alleviated in PS-KQ mice that appeared more similar to the WT control animals. The number of astrocytes in the hippocampus were quantified in B. C, hippocampal CA1 images are shown depicting Iba1-positive microglia (red), which are also reduced in the PS-KQ mice as quantified in D. E, we note a distinct disease-associated amoeboid morphology in PS19 mice, whereas PS-KQ were enriched with ramified microglia, as quantified in F (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.005; ns ϭ not significant). Error bars represent Ϯ S.E. Scale bar ϭ 50 m.

A tau K280Q mutation suppresses P301S pathology mice via impaired NMDAR activation and restored synaptic integrity. Discussion
In this study, we evaluated cell culture models and transgenic mice to assess whether an acetylation-mimic K280Q mutation could alter the progression of P301S-induced tauopathy. We found that the PS-KQ double mutant generates a less toxic tau species that is predominantly hypo-phosphorylated, less MC1immunoreactive, concentrated to the neuronal soma, and associated with impaired tau-mediated toxicity.
Previous studies showed that tau K280Q acetylation-mimic altered tau's biochemical properties in several profound ways. First, this mutant impaired tau-mediated MT assembly, as assessed by the reduced ability to bind and stabilize MTs in cultured cells (1). Given the affinity of residue Lys-280 for negatively charged microtubules, acetylation likely reduces tau association with MTs due to charge neutralization. Second, we showed that K280Q could accelerate tau aggregation in vitro (17), which is consistent with a recent study that showed chemical acetylation specifically at Lys-280 enhanced tau aggregation into globular tau oligomers (19). Finally, K280Q mutant tau expression in a transgenic Drosophila model led to fly locomotion defects and neurodegeneration, in which the authors proposed that Lys-280 acetylation exacerbates neurotoxicity (5). These studies provided the rationale for us to evaluate K280Q-induced toxicity in cell models and tau transgenic mice.

Figure 7. Restored synaptic homeostasis in PS-KQ acetylation-mimic mice despite somatodendritic tau re-localization.
A, hippocampal CA1 regions were double-labeled with tauY9 (green) and the dendritic marker MAP2 (red), which labels the somatodendritic neuronal compartment. Both PS19 and PS-KQ mice contained human transgenic tau that accumulated within dendrites (see merged panels in yellow). Scale bar ϭ 50 m. B, immunoblotting of total NR2B, phospho-NR2B at residue Tyr-1472, PSD-95, and GAPDH from cortical and hippocampal lysates showed reduced full-length NR2B, increased NR2B fragmentation of ϳ65 kDa (associated with NR2B activation), and reduced PSD-95 levels in PS19 mice, all of which were completely or partially restored in the PS-KQ mice. The asterisk represents a cross-reactive band detected by the phospho-NR2B antibody. C, quantification of changes in synaptic markers are shown. Statistical significance was assessed using a Student's t test from n ϭ 3 independent biological replicates (*, p Ͻ 0.05; **, p Ͻ 0.01; ns ϭ not significant). Error bars represent Ϯ S.E.

A tau K280Q mutation suppresses P301S pathology
Our initial experiments performed in cultured cells indicated that K280Q, but not a K280R control mutant, led to tau hypophosphorylation (Fig. 1), hinting that acetylation mimics could potentially reduce phosphorylated tau. It is plausible that K280Q-induced dissociation from MTs may lead to a free tau pool that is more accessible to PP2A-mediated dephosphorylation at AD-relevant phospho-tau epitopes, including Ser-202/ Thr-205, Thr-231, Ser-396, and Ser-404 ( Fig. 1 and Fig. S1A). In light of these findings in vitro, we sought to address the toxicity of this mutant in vivo. Indeed, a similar extent of tau dephosphorylation was observed in PS-KQ mice compared with parental PS19 mice (Fig. 3). We note there was a trend toward reduced PS-KQ tau expression compared with PS19 tau. Although expression differences could account for the observed phenotypes, the points below argue against this possibility. First, PS-KQ tau expression was widespread in all brain regions analyzed by IHC (Fig. 2C). Second, PS-KQ tau was strongly immunoreactive in the somatodendritic compartment at comparable or even slightly higher levels than that of PS19 mice (Fig. 7A). Lastly, our follow-up experiments using cellbased assays support an alternative model in which PS-KQ tau generates a rapid, stable change in tau conformation that inhibits the formation of pathological tau that forms the MC1 and tauC3 epitopes (Fig. 5).
It is intriguing that the K280Q mutation accelerated tau aggregation using in vitro-purified tau models (17) but failed to enhance tau pathology in PS-KQ-expressing cells and mice in this study. We describe four scenarios to explain these findings.
First, additional factors in vivo likely control tau stability and aggregation, including quality control mechanisms such as autophagy and proteasome-mediated degradation. Although K280Q is aggregate-prone in vitro, compensatory factors may become activated in vivo and target PS-KQ tau for enhanced degradation. Indeed, hyper-phosphorylated toxic forms of PS-KQ tau were specifically depleted in mice (Fig. 3). The selective targeting and degradation of phosphorylated PS-KQ tau could occur via activation of the quality control machinery such as the ubiquitin ligase CHIP, previously shown to ubiquitinate and target phospho-tau for degradation (34). During human AD progression, however, impairments in proteostasis and quality control may allow acetylated tau variants to accumulate, which could promote tau aggregation and enhance toxicity, but only in a more susceptible diseased environment.
Second, the biochemical properties of PS-KQ could be directly related to the combination of mutations at residues Pro-301 and Lys-280. PS-KQ mice harbor an aggregate-prone P301S mutation in addition to the K280Q mutation, yielding a double-mutant tau species. It is plausible that K280Q may have quite distinct phenotypes in the context of WT versus P301S transgenic backgrounds. In fact, there is an interesting precedence for opposing phenotypes in different tau backgrounds. For example, under conditions where the Pin1 isomerase was overexpressed, tauopathy was suppressed in WT tau transgenic mice but exacerbated in tau PS19 transgenic mice (35). Therefore, whether the particular tau species contains a WT versus P301S (or P301L) mutation could determine the modifier effects of the additional K280Q mutation. Although additional studies are warranted, our results in seeded cells hint that expo-sure to tau seeds may increase the pool of phosphorylated (single mutant) K280Q tau in both soluble and insoluble fractions (Fig. 5H, compare lanes 1-3 and lanes 7-9), whereas the identical tau seeds reduced PS-KQ phosphorylation (double mutant) (Fig. 5H, compare lanes 4 -6 and lanes 10 -12). Thus, these data suggest K280Q-induced toxicity, but alleviation of toxicity in the context of the double mutant P301S/K280Q, the latter of which may occur via a conformationally distinct tau species.
Third, the K280Q mutation is a charge neutralization mimic that acts as a surrogate for Lys-280 acetylation. It is plausible that K280Q may not fully recapitulate the effects of tau acetylation at residue Lys-280 in vivo. We recently showed that the deacetylase HDAC6 docks onto tau in vulnerable neurons and may act in a compensatory capacity to suppress acetylated tau accumulation (36). The ability of HDAC6 to physically engage and recognize acetylated tau may be required for efficient tau trafficking along neuronal processes to neuritic beads, sites of tau accumulation that contain activated NMDARs and excessive intracellular calcium accumulation (36). Because HDAC6 may not engage acetylation-mimic tau in quite the same manner as acetylated tau, this could result in inefficient tau binding, reduced trafficking, and reduced toxicity. Under this scenario, acetylation-mimic tau mutations may not fully recapitulate tau acetylation. Therefore, manipulating the enzymes that control tau acetylation (e.g. tau HATs and HDACs) may be necessary to further address the impact of global tau acetylation (37).
Finally, in addition to HDAC6 binding, it is plausible that the constitutive mutation K280Q could perturb tau interactions with other neuronal factors required for tau transport and/or tau triage. For example, several chaperones, including Hsp27 (38), Hsc70 (39), Hsp70 (40,41), Hsp90 (42), Hsp110 (43), and FKBP12 and Pin1 family members (44), have all been implicated in tau triage, many of which physically associate with tau via the MTBR domain (39), which harbors most of the acetylated lysine residues. Hsc70 in particular may be critical for packaging and transporting tau complexes via slow axonal transport (45,46). Furthermore, chaperone inhibition has been linked to neuroprotection against tau-mediated toxicity (47)(48)(49)(50). It remains unclear whether altered tau-chaperone interactions are indeed observed in the PS-KQ mice. However, such a scenario could be consistent with reduced PS-KQ processing and reduced synaptotoxicity. Studies are ongoing to address whether dissociation of chaperones from PS-KQ may explain the reduced pathology and neurodegeneration observed in these mice.
Regardless of the exact mechanism, our results suggest that PS-KQ generates a distinct tau conformation that is unique from the toxic P301S variant, shows impaired ability to generate an aggregation-competent tau conformation (e.g. MC1-immunoreactive), and is incapable of inciting excitotoxic neuronal damage. We would like to explicitly state that our findings do not confirm or negate a role for tau acetylation as a driver or suppressor of tau pathology. Rather, our data suggest that manipulating a critical residue in the 2nd MTBR repeat (Lys-280) is capable of significantly altering tau's properties. Additional studies are needed to firmly establish a role for acetyl A tau K280Q mutation suppresses P301S pathology group transfer onto residue Lys-280 as a bona fide modulator of tau pathology in vivo.
In summary, aberrant tau species can potentially be manipulated to alter tau conformation and suppress tau-mediated toxicity. Given that tau strains comprising distinct conformers have been implicated in AD and a range of tauopathies (51,52), it is plausible that therapeutic strategies to target specific tau residues within the MTBR in a site-selective manner (e.g. using CRISPR technology) could represent an avenue to detoxify pathogenic tau strains. This approach could be used to slow neurodegeneration in human patients.

Plasmids and cell culture
The human tau constructs used in these experiments comprised full-length tau containing one or two N-terminal inserts and all four repeat domains (1N4R-tau or 2N4R-tau, also designated as tau-T34 or tau-T40, respectively). The tau plasmids were cloned into the pCDNA5/TO vector (Life Technologies, Inc.). The K280Q, K280R, and P301L (or P301S) tau mutations were generated using site-directed mutagenesis (New England Biolabs). QBI-293 cells (Thermo Fisher Scientific, catalog no. R70507) are commercially available and were maintained according to standard protocols. These cells are a subclone of the standard HEK-293 line with a relatively flat morphology, grow more slowly, and maintain ectopic plasmid expression at more physiological levels compared with the parental line.
-Phosphatase assay QBI-293 cells were grown and transfected with the tau mutants as mentioned above. Cells were lysed in 1ϫ RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 1% Igepal CA-630, 0.1% SDS) without phosphatase inhibitors. Protein phosphatase reactions and control reactions were performed according to the manufacturer's instructions (New England Biolabs catalog no. P0753S). Briefly, reaction mixtures of 10 l of cell lysate, 2 l of 10ϫ New England Biolabs buffer, 2 l of MnCl 2 , and 1 l of -phosphatase per sample were incubated for 1 h at 30°C. The pair of samples for each protein (with and without phosphatase treat-ment) was run side-by-side to enable direct comparison of migration by Western blotting.

tau fibril-induced seeding assay
QBI-293 cells were grown as mentioned above followed by transduction with or without P301L (PL) tau fibrils composed of the microtubule repeat fragment spanning residues 244 -372 (referred to as the tau-K18 fragment) in 6-well plates. The tau fibrils were prepared by resuspending pellets from tau-K18 -PL fibril reactions in OptiMEM (Gibco) followed by sonication (OptiMEM buffer was used as no fibril control). Subsequently, 6 g of sonicated tau-K18 -PL fibrils (or fibril controls) were then combined with Lipofectamine 2000 (Invitrogen) and allowed to incubate at room temperature for 20 min, and the tau fibril/Lipofectamine mixture was added to fulllength tau-expressing cells. After an overnight incubation, cell culture media were replaced with DMEM. Cells were harvested after 48 h using Triton X-100 lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.6) supplemented with deacetylase, phosphatase, and protease inhibitors (3 M TSA, 10 mM NCA, and 1 mM NaF; 1 mM Na 3 VO 4 ; 1 mM PMSF; and protease inhibitor mixture) to generate the soluble fraction. The pellet was resuspended in SDS lysis buffer (1% SDS, 150 mM NaCl, 50 mM Tris, pH 7.6), supplemented with phosphatase and protease inhibitors as mentioned above, to generate insoluble fractions.

tau transgenic mouse models
The tau PS19 transgenic mice that express P301S mutant human microtubule-associated protein tau driven by the mouse prion protein promoter are well-characterized and have been previously described (22). The transgene encodes the disease-associated P301S mutation and includes four microtubule-binding domains and one N-terminal insert (1N4R). The PS-KQ mice were generated at the UNC animal core facility by incorporating a single acetylation-mimicking mutation in the tau transgene at Lys-280 (K280Q). The transgene DNA was injected into the pronuclei of C57BL/6 embryos and implanted into pseudo-pregnant recipient females. Three founder lines (referred to as PS-KQ lines 6, 12, and 13) were generated. After initial characterization of tau, PS-KQ line 13 mice were used throughout this study, with all experiments confirmed in parallel using PS-KQ line 12. Males and females of both transgenic and WT lines were used in a balanced manner for all biochemical and histology experiments. There were no differences in transgene expression or extent of pathology in male and female PS-KQ mice. This study was performed in strict compliance with animal protocols approved by the Institutional Animal Care and Use Committees (IACUC) of the University of North Carolina at Chapel Hill under an approved protocol.

Biochemical extraction of mouse brain
Fractionation of mouse brain was performed by sequential extraction using buffers of increasing strength. Mouse brain tissue was homogenized in 4 volumes per g of high-salt buffer (10mM Tris base, 500mM NaCl and 2mM EDTA) supplemented with deacetylase, phosphatase and protease inhibitors (2 M TSA, 10 mM NCA, 1 mM NaF; 1 mM Na3VO4; 1 mM PMSF; and protease inhibitor cocktail) and centrifuged at 15,000 ϫ g for 45 A tau K280Q mutation suppresses P301S pathology min to generate high-salt fractions. Resulting pellets were reextracted in 4 volumes per g of RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS). Myelin flotation was performed on pellets re-extracted in RIPA buffer supplemented with 20% sucrose. Finally, resultant insoluble pellets were extracted in 1 volume per g of tissue in SDS buffer (1% SDS in 50 mM Tris, 150 mM NaCl, pH 7.6). The tau protein levels in the soluble and insoluble fractions were analyzed by SDS-PAGE and Western blotting using the indicated antibodies to detect total or phosphorylated tau proteins.

Immunofluorescence (IF) microscopy
Mice were deeply anesthetized and transcardially perfused with 15 ml of phosphate-buffered saline (PBS), and brains were removed, followed by immersion fixation for 48 h with 10% formalin in 1ϫ PBS and paraffin embedding. Paraffin blocks were sectioned at 5-m thickness for IF and IHC analyses. Briefly, sections were deparaffinized and hydrated to dH 2 O, immersed in 1ϫ PBS, pH 7.4, for 1 h. Antigen retrieval was done by boiling sections in citrate buffer, pH 6.0; endogenous peroxidases were quenched with 5% H 2 O 2 in water for 30 min, and sections were blocked in 0.1 M Tris with 2% fetal bovine serum for 5 min. Sections were then incubated with primary antibody overnight at 4°C. IF analyses were performed using Alexa Fluor 488-and 594-conjugated secondary antibodies (Molecular Probes). IF analyses were performed using Alexa Fluor 488-and 594-conjugated secondary antibodies (Molecular Probes). An Olympus IX83 inverted microscope was used to analyze all of the mouse tissues. Statistical analysis was determined using a two-tailed unpaired Student's t test with unequal variance (significance set as p Ͻ 0.05). All quantitative fluorescence were independently validated with a minimum of three different replicates. Primary antibodies used for IF were Iba1 (WAKO 019-19741 1:500), GFAP (Abcam ab53554, 1:500), MAP2 (Millipore, AB5622, 1:500), and TauY9 (Enzo BML-TA3119-0100, 1:1000).
Thioflavin-S (ThS) (Sigma) staining was performed as follows. Sections were deparaffinized and hydrated to dH 2 O as mentioned above, followed by antigen retrieval. Sections were then rinsed in PBS, immersed in 0.05% KMnO 4 /PBS for 4 min, and destained in 0.2% K 2 S 2 O 5 , 0.2% oxalic acid in PBS, immersed in 0.0125% ThS dissolved in 40% EtOH/60% PBS and differentiated in 50% EtOH/50% PBS for 10 -15 min. After differentiation, sections were treated for autofluorescence with Sudan Black solution for 30 s and quickly rinsed with 70% EtOH followed by washing with dH 2 O, incubation with DAPI, and cover-slipped.
Cell culture IF experiments were performed using QBI-293 cells grown on poly-D-lysine-coated coverslips and transfected with 1N4R tau constructs (tau-T34) containing WT, P301S, K280Q, or K280Q/P301S mutants for 48 h. Cells were fixed in 4% paraformaldehyde for 15 min, rinsed three times in PBS, and permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 10 min. Cells were then blocked in 2% milk for 1 h and incubated overnight at 4°C with the primary antibodies described above. Cells were washed in PBS and incubated with Alexa 488-or Alexa 594 -conjugated secondary antibodies and analyzed using the Olympus IX83 inverted microscope.

IHC analysis
IHC analysis was performed using the avidin-biotin complex detection system (Vector Laboratories) and 3,3Ј-diaminobenzidine. Briefly, sections were deparaffinized and rehydrated, and antigen retrieval was performed by boiling sections in citrate buffer, pH 6.0. Endogenous peroxidases were quenched with 5% H 2 O 2 in water for 30 min, and sections were blocked in 0.1 M Tris with 2% fetal bovine serum for 5 min. Primary antibodies were incubated overnight at 4°C. After washing, sections were sequentially incubated with biotinylated secondary antibodies for 1 h and avidin-biotin complex for 1 h. Bound antibody complexes were visualized by incubating sections in DAB substrate (Sigma, D3939), a solution containing 100 mM Tris-HCl, pH 7.6, 0.1% Triton X-100, 1.4 mM diaminobenzidine, 10 mM imidazole, and 8.8 mM H 2 O 2 . Sections were then lightly counterstained with hematoxylin, dehydrated, and cover-slipped. The entire cortex and hippocampus was imaged using a Nikon EclipseTi2 inverted microscope(ϫ20) (Fig. S2).

Statistical analyses
GraphPad Prism software was used for all statistical analyses. Results were polled from a minimum of n ϭ 3 independent experiments and presented as average Ϯ standard error of the mean (S.E.). Comparisons between the two groups were analyzed using unpaired Student's t test. Kaplan-Meier survival curves were calculated using WT (n ϭ 22), PS19 (n ϭ 27), and PS-KQ (n ϭ 25), and statistical significance was determined by the log-rank (Mantel-Cox) test (****, p Ͻ 0.0001).