Abstract

In Alzheimer's disease, tau is predominantly acetylated at K174, K274, K280, and K281 residues. The acetylation of K274-tau is linked with memory loss and dementia. In this study, we have examined the molecular mechanism of the toxicity of acetylated K274-tau. We incorporated an acetylation mimicking mutation at K274 (K→Q) residue of tau. The mutation (K274Q) strongly reduced the ability of tau to bind to tubulin and also to polymerize tubulin while K274R mutation did not reduce the ability of tau either to bind or polymerize tubulin. In addition, K274Q-tau displayed a higher aggregation propensity than wild-type tau as evident from thioflavin S fluorescence, tryptophan fluorescence, and electron microscopic images. Furthermore, dynamic light scattering, atomic force microscopy, and dot blot analysis using an oligomer-specific antibody suggested that K274Q mutation enhanced the oligomerization of tau. The K274Q mutation also strongly decreased the critical concentration for the liquid–liquid phase separation of tau. The oligomeric forms of K274Q-tau were found to be more toxic than wild tau to neuroblastoma cells. Using circular dichroism and fluorescence spectroscopy, we provide evidence indicating that the acetylation mimicking mutation (K274Q) induced conformational changes in tau. The results suggested that the acetylation of tau at 274 residues can increase tau aggregation and enhance the cytotoxicity of tau oligomers.

Introduction

Alzheimer's disease (AD) causes gradual degeneration of neurons associated with the synaptic deterioration and memory loss. AD and frontotemporal dementia (FTD) are mainly characterized by the formation of neurofibrillary tangles composed of hyperphosphorylated tau. However, tau hyper-phosphorylation alone is not responsible for the cognitive decline and neuronal dysfunction associated with the AD and FTD [1,2]. Tau acetylation is also reported to be a major factor involved in tauopathies [36]. The level of acetylated tau is increased in Alzheimer's brain and in other tauopathies, which leads to cognitive decline and neurodegeneration [3,7,8]. Tau gets acetylated predominantly on microtubule-binding region (MTBR) by p300, which is found to be elevated in the AD [3,7,9,10]. The acetylation of tau inhibits the binding of tau to microtubules [7], increases microtubule dynamics [11], enhances tau accumulation, and promotes the aggregation of tau in neurons [3,12].

In Alzheimer's brain, tau is acetylated mostly at K280 [5], K281 [4,8], K274 [4,8], and K174 [12] residues. LC–MS-MS analysis of tau isolated from human Alzheimer's brain and rTg4510 mouse brain revealed that tau is predominantly acetylated at K274 residue [8]. The incorporation of the K274 and K281 acetylation mimicking mutant tau into transgenic mice was reported to increase the tau pathogenicity by reducing the neuronal plasticity and obstructing AMPAR trafficking [8]. The acetylation of tau at Lys 274 was linked to dementia [8]. The enhancement of Ac-274 and Ac-281 tau was indicated to reduce the memory-related protein KIBRA in the synapse and to arrest the activity-dependent F-actin assembly which results in the inhibition of AMPAR insertion and synapse potentiation [8]. Tau also interacts with F-actin and regulates its bundling [1316]. Therefore, it is possible that the acetylation at K274 and K281 residues might decrease the interaction of F-actin and tau leading to the disassembly of F-actin during activity-dependent polymerization [8]. The expression of acetylation mimicking K274/281 tau in mice decreased the level of cytoskeletal protein AnkG and βIV-spectrin, destabilized the axon and caused somatodendritic mislocalization of tau [11]. Furthermore, it increased the microtubule dynamics in neuronal cells [11]. The effect of ac-K280 and ac-K281 on tau-microtubule interactions, tau aggregation was studied earlier [17]. However, the effect of K274 acetylation on tau at the molecular level is not clear. The consequences of the acetylation at K274 residue of tau on the interaction with microtubules and microtubule polymerization are unknown. Also, a little information is available about the aggregation properties and toxicity of Ac-K274-tau.

However, it is extremely difficult to acetylate a specific residue in a protein in vitro. To overcome this problem, an acetylation mimicking (K→Q) mutation is widely used for tau [7,8,17] and for several other proteins such as histone [18,19] and p53 protein [20,21]. The use of acetylation mimicking mutation is a well-established strategy to study the protein acetylation [8,11,17,19]. Therefore, we introduced acetylation mimicking (K→Q) and a non-acetylation mimicking (K→R) mutations at the K274 position of the full-length tau. The acetylation mimicking mutation (K274Q) was found to decrease the ability of tau to polymerize tubulin and to impair the binding of tau to microtubules. In addition, the mutation (K274Q) reduces the ability of tau to induce microtubule bundling in neuroblastoma cells. Furthermore, the acetylation mimicking mutation enhanced the aggregation, oligomerization, and liquid–liquid phase separation (LLPS) of tau. In addition, the oligomers of K274Q-tau were more toxic to neuroblastoma cells than wild-type and K274R-tau indicating that the acetylation at K274 residue of tau plays an important role in the functioning of tau.

Material

Guanosine triphosphate (GTP), isopropyl β-d-1-thiogalactopyranoside (IPTG), phosphocellulose resin (PC resin), poly(A) RNA, thioflavine S (ThS), glutaraldehyde, 1-anilinonaphthalene-8-sulfonic acid (ANS), 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), sodium acetate, polyethylene glycol 8000 (PEG 8000), uranyl acetate, and tau-5 antibody were ordered from Sigma–Aldrich (St Louis, MO, U.S.A.). Dpn I, T4 DNA ligase, A11 oligomer-specific antibody, and chemiluminescent kit were ordered from Thermo Fisher Scientific (Rockford, U.S.A.). Anti-rabbit IgG conjugated with HRP and Bradford's reagent was ordered from Bio-Rad. The KAPA Hi-Fi polymerase was obtained from KAPA Biosystems. All other chemicals were purchased from HiMedia (Mumbai, India). The clone of full-length tau was a gift from Prof. Stuart Feinstein (University of California, Santa Barbara).

Methods

Generation of tau mutants

The K274Q, K274R, Y310W, and K274Q-Y310W (double mutant) mutants of 4R2N tau were generated using KAPA Hi-Fi polymerase. Then amplified templates were treated with Dpn I to remove non-mutated templates. Furthermore, mutated templates were ligated using T4 DNA ligase and transformed into DH5α cells. The mutations were confirmed by DNA sequencing. The details of the primers used for the site-directed mutagenesis are shown in Supplementary Table S1.

Expression and purification of tau and tubulin

Different tau proteins were expressed in E. coli Bl-21 RIPL cells and the proteins were purified by boiling the cell lysate followed by phosphocellulose chromatography using a gradient of NaCl as described earlier [22,23]. The purity of tau was determined to be ≥95% by Coomassie staining of the SDS–PAGE. The protein concentration was measured at 278 nm as explained earlier [23]. Tubulin was purified from goat brain as explained earlier [24] and the concentration of tubulin was determined by the Bradford method [25].

Tubulin polymerization assay

Tubulin (12 µM) in PEM buffer (pH 6.8) was incubated without and with either 3 µM wild-type tau (WT-tau), K274Q-tau or K274R-tau. Subsequently, 1 mM GTP was added into the reaction mixture and the assembly of tubulin was monitored at 37°C using 90° light scattering at 350 nm using a fluorescence spectrophotometer (JASCO FP-6300, Tokyo, Japan) for 30 min.

Sedimentation assay

Tubulin (12 µM) was incubated without and with either 3 µM WT-tau, K274Q-tau or K274R-tau in the presence of 1 mM GTP at 37°C for 30 min. Then, 50 µl fraction was taken out from each sample for loading control. Then, the samples were centrifuged at 26 234 g for 30 min and the supernatant and pellet fractions were separated. Then, the supernatant, pellet, and loading control (total protein) were analyzed by SDS–PAGE. The band intensity was measured using ImageJ software.

Tau aggregation assay

WT-tau, K274Q-tau, or K274R-tau (2 µM) in 100 mM sodium acetate buffer (pH 7.0) was incubated without or with 2 µM heparin or 480 µg/ml poly(A) RNA at 37°C in an orbital shaker for 24 h. The kinetics of tau aggregation was monitored by incubating tau with heparin for different (0, 2, 6, 8, 24, and 72 h) durations. In all the aggregation reactions, the concentration of tau and heparin was used in 1:1 ratio. The aggregation was monitored by thioflavin S assay. Thioflavin S (20 µM) was added to the reaction mixture and the fluorescence intensity of the mixtures was measured using 440 and 521 nm as the excitation and emission wavelengths, respectively.

Dot blot assay

WT-tau (12 µM) and K274Q-tau were incubated with and without heparin for 6 h. The samples were blotted on PVDF membranes. The membrane was treated with 5% skim milk in TBST (Tris-buffered saline with Tween 20 [pH 7.6]) for 2 h and subsequently, incubated with A11 oligomer-specific antibody that detects oligomers of tau [26,27], amyloid β [28] and α-synuclein [29], for 2 h at 25°C followed by incubation with anti-rabbit secondary antibody conjugated with HRP (Bio-Rad) for 1 h at 25°C. Furthermore, the blot was washed with TBST and developed using a chemiluminescent substrate (Thermo Fisher Scientific).

Electron microscopy

Tubulin (12 µM) was incubated with and without 3 µM of wild or K274Q-tau, in the presence of 1 mM GTP at 37°C for 30 min. Tubulin polymers were fixed with 0.5% glutaraldehyde. Then, the polymers were diluted in warm PEM buffer, adsorbed on 200 mesh copper grid for 1 min and stained with 2% uranyl acetate. The polymers were observed under a transmission electron microscope (JEOL JEM-1220) at 120 kV.

WT-tau and K274Q-tau (2 µM) in 100 mM sodium acetate buffer (pH 7.0) was incubated without or with 2 µM heparin at 37°C under shaking condition in an orbital shaker for 24 h. The tau filaments were fixed with 0.5% glutaraldehyde and EM samples were prepared as mentioned above. The length of tau filament was measured using ImageJ software.

Atomic force microscopy

WT-tau and K274Q-tau (2 µM) were incubated without or with 2 µM heparin at 37°C for 6 h under shaking conditions. The samples were fixed with 0.5% glutaraldehyde, adsorbed on freshly cleaved mica discs for 10 min and then, washed with Milli-Q water. The samples were observed by Asylum MFP-3D Bio-AFM.

Dynamic light scattering

The size of soluble and oligomeric tau was measured by dynamic light scattering as described previously [22]. Briefly, WT-tau (12 µM) and K274Q-tau were incubated with and without 12 µM heparin for 6 h at 37°C under shaking condition. Then, the samples were equilibrated at 25°C for 5 min. The hydrodynamic diameter of the sample was measured by DLS particle size analyzer (Zetasizer Pro) from three independent experiments with 25 runs each.

Circular dichroism (CD) spectroscopy

WT-tau (5 µM) or K274Q-tau or K274R-tau was incubated in 5 mM PIPES buffer (pH 6.8). The far-UV (190–250 nm) CD spectra were recorded using 1 mm path length quartz cuvette in a spectropolarimeter (JASCO 1500, Tokyo, Japan). Each spectrum reported was an average of five scans. The experiments were repeated five times. The CD spectra were deconvoluted using Jasco protein secondary structure analysis version 2.1.1.1.

ANS fluorescence

WT-tau, K274Q-tau, or K274R-tau (5 µM) were incubated with 10 µM ANS in PEM buffer (pH 6.8) for 10 min. The emission spectra (425–600 nm) of the mixtures were monitored using 370 nm as the excitation wavelength.

Tryptophan fluorescence

A tryptophan residue was incorporated in both WT-tau and K274Q-tau by substituting Y310 residue with a tryptophan residue. The tryptophan fluorescence of soluble tau and aggregated tau was monitored on a fluorescence spectrophotometer. The emission spectra (310–400 nm) were recorded by exciting the reaction mixture at 295 nm.

DIC microscopy

The LLPS of tau was monitored using the DIC microscopy [30]. WT-tau and K274Q-tau (5 µM) were incubated either with heparin (5 µM) or PEG 8000 (10%) or with both heparin (5 µM) and PEG 8000 (10%) for 2 h. Then, 30 µl of protein sample was placed on a glass slide and DIC images were acquired using a Yokogawa CSU-X1 microscope at 63× oil immersion objective.

Surface plasmon resonance

The binding kinetics of WT-tau, K274Q-tau, and K274R-tau with tubulin was measured by surface plasmon resonance (SPR) using Biacore 2000 in three separate experiments. Tubulin (250 µg/ml) was immobilized on CM5 chip using amine coupling method. The blank immobilization was done in flow cell 1 while tubulin was immobilized to 3000RU in flow cell 2. Then, different concentrations of WT-tau, K274R-tau, and K274Q-tau were injected with a flow rate of 40 µl/ml through both flow cells. The HBSE-P (10 mM HEPES, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.005% P-20) with pH 7.4 was used as running buffer. Regeneration was done using 10 mM glycine buffer (pH 2.5). The binding sensorgram of tau and tubulin (flow cell 2) was subtracted with tau sensorgram (flow cell 1). Then obtained sensorgram was fitted into 1:1 binding model and rate constants (Kon and Koff) were calculated using Biacore T200 Evaluation Software. The sets which pass the quality control test of Biacore T 2000 software were selected for Kd calculation.

Alamar blue assay

Tau (20 µM) was incubated with 20 µM heparin in sodium acetate buffer (pH 7.0) for 6 h at 37°C under shaking condition. After 6 h incubation, both WT-tau and K274Q-tau formed oligomers. The oligomers were collected through centrifugation at 55 000 rpm. Then, the sedimented oligomers were suspended in PBS buffer and the concentration was measured at 280 nm. SH-SY5Y cells were cultured as described earlier [31]. SH-SY5Y cells were grown without or with different concentrations of soluble and oligomeric WT, K274Q, and K274R-tau for 48 h in 96-well plate. Then, the cells were washed and 10 µg of the sterile alamar blue solution was added in each well and incubated for 1 h at 37°C in a CO2 incubator. Fluorescence emission at 590 nm was recorded after excitation at 570 nm in SpectraMax M2e. Cell viability was calculated as described earlier [32].

Microtubule staining

To check the effect of soluble tau on the microtubule integrity in neuroblastoma cells, we incubated SH-SY5Y cells with 5 µM of soluble WT-tau and K274Q-tau for 48 h on a coverslip. Then, the cells were fixed with 4% formaldehyde, permeabilized by 100% methanol, and incubated with 2% BSA for 1 h at room temperature. The cells were incubated with α tubulin antibody raised in mouse for 16 h at 4°C. The unbound antibody was washed out with PBS. Subsequently, the cells were stained with anti-mouse IgG tagged with FITC. Hoechst 33258 was used to stain DNA. The images were acquired in a Yokogawa CSU-X1 microscope at 63× oil immersion objective.

Internalization of oligomers in cells

To check the internalization of tau oligomers into the neuroblastoma cells, we incubated SH-SY5Y cells with 5 µM of tau oligomers for 48 h on a coverslip. Then, the cells were fixed with 4% formaldehyde, permeabilized by 100% methanol, and incubated with 2% BSA for 1 h at room temperature. The cells were incubated with an A11 oligomer-specific antibody [2629] raised in rabbit for overnight at 4°C. The unbound antibody was washed out with PBS. The cells were then incubated with a tau-5 antibody raised in the mouse for 3 h at 25°C. Subsequently, the cells were stained with anti-rabbit IgG tagged with Alexa 594 and anti-mouse IgG tagged with FITC. Hoechst 33258 was used to stain DNA. The images were acquired in a Yokogawa CSU-X1 microscope at 63× oil immersion objective. The percentage of tau colocalizing with A11 oligomers staining was determined using JACoP algorithm from ImageJ software and the Pearson's correlation coefficient of colocalization was determined by scoring 126 cells [33].

Statistical analysis

The graphs were plotted using GraphPad Prism 6.0. The results were analyzed using a t-test where the results with a P-value <0.05 were taken as significant.

Results

The K274Q mutation changed the secondary structure of tau

Tau is an intrinsically disordered protein with a minimal secondary structure content where 274KVQIINKKLDL284 (R2) and 305SVQIVYKPVDL315 (R3) region have the highest amount of β sheet [2,34]. There are 23 lysine residues of tau which are acetylated by p300 acetyltransferase [3]. Most of these residues lie in the MTBR region of tau [3]. Since the K274Q mutation is in the R2 region of the MTBR domain of tau, we checked the effect of the K274Q mutation on the secondary structures of tau. The secondary structures of WT-tau, K274R-tau, and K274Q-tau were analyzed by monitoring the far-UV circular dichroism spectra of the proteins. The ratio of 222/208 for WT-tau and K274R-tau was determined to be 0.4 ± 0.003 and 0.41 ± 0.03, respectively, indicating that the non-acetylation mimicking mutation K274R does not show any change in the secondary structure of tau (Figure 1A,B). However, the 222/208 ratio was increased to 0.57 ± 0.07 for K274Q-tau, which was significantly different (P-value < 0.001) from that of the WT-tau indicating that the acetylation mimicking mutation at K274 altered the secondary structure of tau (Figure 1A,B).

K274Q mutation changes the secondary structure of tau.

Figure 1.
K274Q mutation changes the secondary structure of tau.

(A,B) Circular dichroism spectra of WT-tau (O), K274R-tau (Δ), and K274Q-tau (●) are shown. (B) The graph showing the 222/208 ratio of WT-tau, K274R-tau, and K274Q-tau (***P-value < 0.001). (C) The fluorescence spectra of ANS with WT-tau (●), K274R-tau (), and K274Q-tau (▴) (***P-value < 0.001). (D) The fluorescence spectra of Y310W tau (●) and double mutant tau (K274Q and Y310W) () are shown (***P-value < 0.001). The experiment was performed five times.

Figure 1.
K274Q mutation changes the secondary structure of tau.

(A,B) Circular dichroism spectra of WT-tau (O), K274R-tau (Δ), and K274Q-tau (●) are shown. (B) The graph showing the 222/208 ratio of WT-tau, K274R-tau, and K274Q-tau (***P-value < 0.001). (C) The fluorescence spectra of ANS with WT-tau (●), K274R-tau (), and K274Q-tau (▴) (***P-value < 0.001). (D) The fluorescence spectra of Y310W tau (●) and double mutant tau (K274Q and Y310W) () are shown (***P-value < 0.001). The experiment was performed five times.

A hydrophobic fluorescent molecule, ANS, is widely used to determine the hydrophobic surface arrangements in proteins [35,36]. The fluorescence intensity of ANS was found to be 22 ± 2% higher in the presence of K274Q-tau than both WT-tau and K274R-tau indicating that the mutation (K274Q) induced a conformational change in tau (P-value < 0.001) (Figure 1C). Furthermore, we checked whether the K274Q mutation could induce a conformational change in tau using tryptophan fluorescence. Tau lacks a tryptophan residue; therefore, a tryptophan residue was introduced in both WT-tau and K274Q-tau through a site-directed mutation (Y310W). The tryptophan fluorescence of K274Q-tau was found to be 67 ± 5% lesser than WT-tau (P-value < 0.001) (Figure 1D). The results together suggested that the K274Q mutation in tau perturbed the conformation of the protein.

K274Q-tau bound to tubulin with a lower affinity than WT-tau

An elevated level of free tau was suggested to increase the aggregation of tau [3739]. Thus, the binding of tubulin to tau can reduce tau aggregation by decreasing the concentration of free tau. In addition, the binding of tau to tubulin may enhance tubulin assembly. Therefore, we examined the effect of the K274Q mutation in tau on its ability to bind to tubulin and to promote tubulin assembly. The binding of tau to tubulin was examined using SPR. The dissociation constant of the interaction of tubulin with K274Q-tau and WT-tau was determined to be 1.5 ± 0.2 and 0.7 ± 0.03 µM, respectively, indicating that K274Q-tau has a less affinity (P-value < 0.01) towards tubulin than WT-tau (Figure 2A,B and Supplementary Table S2). Furthermore, the Kon rate for WT-tau and K274Q-tau was determined to be 10961 ± 802 and 7342 ± 980 M−1 S−1 (P-value < 0.01), respectively (Supplementary Table S2). The Kon rate suggested that the WT-tau binds more quickly to tubulin than K274Q-tau. The Koff rate for WT-tau and K274Q-tau was estimated to be 0.009 ± 0.0012 and 0.01 ± 0.0016 S−1, respectively (Supplementary Table S2). The binding analysis indicated that the binding affinity of K274R-tau for tubulin was similar to that of WT-tau while the acetylation mimicking mutation at the K274 position of tau reduced the binding affinity of tau towards tubulin (Figure 2C and Supplementary Table S2).

K274Q mutation of tau decreases the binding of tubulin and tau.

Figure 2.
K274Q mutation of tau decreases the binding of tubulin and tau.

The sensorgram showing the binding kinetics of tubulin with 0.039 (●), 0.078 (▪), 0.156 (▴), 0.312 (▾), 0.625 (♦), 1.25 (o), 2.5 (□), 5 (Δ), 10 (∇) µM WT-tau (A), 0.039 (●), 0.078 (▪), 0.156 (▴), 0.312 (▾), 0.625 (♦), 1.25 (o), 2.5 (□), 5 (Δ), 10 (∇) µM K274Q-tau (B), and 0.039 (●), 0.078 (▪), 0.156 (▴), 0.312 (▾), 0.625 (♦), 1.25 (o), 2.5 (□), 5 (Δ), 10 (∇) µM K274R-tau (C). The experiment was repeated six times.

Figure 2.
K274Q mutation of tau decreases the binding of tubulin and tau.

The sensorgram showing the binding kinetics of tubulin with 0.039 (●), 0.078 (▪), 0.156 (▴), 0.312 (▾), 0.625 (♦), 1.25 (o), 2.5 (□), 5 (Δ), 10 (∇) µM WT-tau (A), 0.039 (●), 0.078 (▪), 0.156 (▴), 0.312 (▾), 0.625 (♦), 1.25 (o), 2.5 (□), 5 (Δ), 10 (∇) µM K274Q-tau (B), and 0.039 (●), 0.078 (▪), 0.156 (▴), 0.312 (▾), 0.625 (♦), 1.25 (o), 2.5 (□), 5 (Δ), 10 (∇) µM K274R-tau (C). The experiment was repeated six times.

K274Q mutation of tau reduced its ability to polymerize tubulin

The effect of K274Q mutation of tau on the polymerization of tubulin was monitored using light scattering, sedimentation assay, and electron microscopy (Figure 3). WT-tau strongly enhanced the rate and the extent of tubulin assembly as indicated by the light scattering assay (Figure 3A,B). The K274Q mutation in tau significantly reduced its ability to polymerize tubulin (P-value < 0.001) while K274R had no visible effect on tubulin assembly (Figure 3A,B). Furthermore, even high concentrations of K274Q-tau were not able to increase the assembly of tau like the WT-tau (Figure 3C,D). In addition, WT-tau and K274R-tau strongly increased the amount of polymerized tubulin as compared with K274Q-tau (Figure 3E,F). The EM analysis also showed dense filaments of microtubules in the presence of WT-tau; in contrast, only a few microtubules per field of view were found in the presence of K274Q-tau (Figure 3G–I). Furthermore, we incubated neuroblastoma cells with either soluble WT-tau or K274Q-tau for 48 h. The bundling of microtubules was observed in the neuroblastoma cells incubated with WT-tau while no microtubule bundle was found in the presence of K274Q-tau (Figure 4). The results suggested that a decrease in the binding affinity of tau to tubulin due to the K274Q mutation in tau decreases its ability to stabilize microtubules.

K274Q mutation of tau reduced its ability to polymerize tubulin.

Figure 3.
K274Q mutation of tau reduced its ability to polymerize tubulin.

(A,B) Tubulin (12 µM) in PEM buffer (pH 6.8) was polymerized with 1 mM GTP in the absence (♦) or presence of either 3 µM WT-tau (O) or K274R-tau (Δ) or K274Q-tau (●). Quantification of polymerization monitored by 90° light scattering assay (n = 4, ***P-value < 0.001) (B). (C,D) Tubulin (12 µM) was polymerized in the absence (Δ) or presence of 1 µM (O), 2 µM (◊), and 3 µM (□) of WT-tau or 1 µM (●), 2 µM (♦), and 3 µM (▪) K274Q-tau (n = 4, *P-value < 0.05). (E,F) Sedimentation assay. Tubulin (12 µM) in PEM buffer (pH 6.8) was polymerized in the presence of 1 mM GTP and 3 µM of WT-tau or K274R-tau or K274Q-tau. Then the pellet fraction (P) and supernatant fraction (S) and 3% of total protein (T) were analyzed by SDS–PAGE. The tau to tubulin band intensity ratio in the total protein of WT and K274Q input was found to be similar (n = 4, ***P-value < 0.001). The results are represented as ±SEM. (G–I) EM analysis of tubulin polymers. Tubulin polymerized in the absence of tau (G) and in the presence of WT-tau (H) and K274Q-tau (I). Scale bar is 200 nm.

Figure 3.
K274Q mutation of tau reduced its ability to polymerize tubulin.

(A,B) Tubulin (12 µM) in PEM buffer (pH 6.8) was polymerized with 1 mM GTP in the absence (♦) or presence of either 3 µM WT-tau (O) or K274R-tau (Δ) or K274Q-tau (●). Quantification of polymerization monitored by 90° light scattering assay (n = 4, ***P-value < 0.001) (B). (C,D) Tubulin (12 µM) was polymerized in the absence (Δ) or presence of 1 µM (O), 2 µM (◊), and 3 µM (□) of WT-tau or 1 µM (●), 2 µM (♦), and 3 µM (▪) K274Q-tau (n = 4, *P-value < 0.05). (E,F) Sedimentation assay. Tubulin (12 µM) in PEM buffer (pH 6.8) was polymerized in the presence of 1 mM GTP and 3 µM of WT-tau or K274R-tau or K274Q-tau. Then the pellet fraction (P) and supernatant fraction (S) and 3% of total protein (T) were analyzed by SDS–PAGE. The tau to tubulin band intensity ratio in the total protein of WT and K274Q input was found to be similar (n = 4, ***P-value < 0.001). The results are represented as ±SEM. (G–I) EM analysis of tubulin polymers. Tubulin polymerized in the absence of tau (G) and in the presence of WT-tau (H) and K274Q-tau (I). Scale bar is 200 nm.

K274Q mutation of tau reduced its ability to induce microtubule bundling in neuroblastoma cells.

Figure 4.
K274Q mutation of tau reduced its ability to induce microtubule bundling in neuroblastoma cells.

SH-SY5Y cells were incubated with 5 µM of soluble tau (WT-tau or K274Q-tau) for 48 h. In the presence of WT-tau, microtubules formed bundles in the cells (shown by arrow) while in the cells incubated with K274Q-tau, microtubules were well spread in the cytoplasm. The scale bar is 10 µm.

Figure 4.
K274Q mutation of tau reduced its ability to induce microtubule bundling in neuroblastoma cells.

SH-SY5Y cells were incubated with 5 µM of soluble tau (WT-tau or K274Q-tau) for 48 h. In the presence of WT-tau, microtubules formed bundles in the cells (shown by arrow) while in the cells incubated with K274Q-tau, microtubules were well spread in the cytoplasm. The scale bar is 10 µm.

The acetylation mimicking mutation at K274 enhanced tau aggregation

The aggregation of tau is a key indicator of several tauopathies [40]. We checked the aggregation propensity of WT-tau, K274Q-tau, and K274R-tau in the presence of polyanionic inducers, heparin (Figure 5A–F) and RNA (Figure 5G–I). These inducers are naturally present in the neuronal cells [41]. In the presence of heparin, the aggregation propensity of K274Q-tau was found to be significantly higher than both WT-tau (P-value < 0.001) and K274R-tau as indicated by thioflavin S assay (Figure 5A,B). In a separate experiment, the aggregation of WT-tau and K274Q-tau in the presence of heparin was monitored using Y310W fluorescence of tau proteins (Figure 5C,D). The tryptophan fluorescence intensity of K274Q-tau was found to be reduced more strongly than WT-tau, indicating that the acetylation mimicking mutation (K274Q) enhanced the aggregation of tau (Figure 5C,D). Furthermore, we checked the rate of aggregation of WT-tau and K274Q-tau in the presence of heparin by thioflavin S assay (Figure 5E) and 90° light scattering (Figure 5F). K274Q-tau displayed a much higher rate of aggregation than WT-tau (Figure 5E,F). In addition, we checked the RNA-induced aggregation of tau by thioflavin S assay (Figure 5G,H) and 90° light scattering assay (Figure 5I). The experiments indicated that the aggregation propensity of K274Q-tau is significantly higher than both WT-tau (P-value < 0.001) and K274R-tau (P-value < 0.001) in the presence of RNA (Figure 5G–I).

K274Q mutation enhanced tau aggregation.

Figure 5.
K274Q mutation enhanced tau aggregation.

(A,B) WT-tau (●), K274R-tau (▴), and K274Q-tau (♦) were incubated with heparin for 24 h and the aggregation of tau was monitored by thioflavin S fluorescence. The symbolic representation of tau without heparin is as follows: WT-tau (o), K274R-tau (Δ), and K274Q-tau (◊) (***P-value < 0.001). (C) Tryptophan fluorescence spectra of Y310W tau (O), double mutant tau (◊), Y310W tau plus heparin (●), and double mutant plus heparin. (♦). (D) The double mutant (K274Q, Y310W) tau has more change in tryptophan fluorescence intensity indicating aggregation than Y310W tau (n = 3, ***P-value < 0.001). (E,F) The aggregation kinetics of WT-tau (●) and K274Q-tau (▪) was monitored by thioflavin S fluorescence (E) and 90° light scattering (F). (G–I) WT-tau (●), K274R-tau (▴), and K274Q-tau (♦) were incubated with RNA for 24 h and the aggregation of tau was monitored by thioflavin S fluorescence (G,H) and 90° light scattering (I). The symbolic representation of tau without RNA is as follows: WT-tau (o), K274R-tau (Δ), and K274Q-tau (◊) (***P-value < 0.001). The results are represented as ±SEM.

Figure 5.
K274Q mutation enhanced tau aggregation.

(A,B) WT-tau (●), K274R-tau (▴), and K274Q-tau (♦) were incubated with heparin for 24 h and the aggregation of tau was monitored by thioflavin S fluorescence. The symbolic representation of tau without heparin is as follows: WT-tau (o), K274R-tau (Δ), and K274Q-tau (◊) (***P-value < 0.001). (C) Tryptophan fluorescence spectra of Y310W tau (O), double mutant tau (◊), Y310W tau plus heparin (●), and double mutant plus heparin. (♦). (D) The double mutant (K274Q, Y310W) tau has more change in tryptophan fluorescence intensity indicating aggregation than Y310W tau (n = 3, ***P-value < 0.001). (E,F) The aggregation kinetics of WT-tau (●) and K274Q-tau (▪) was monitored by thioflavin S fluorescence (E) and 90° light scattering (F). (G–I) WT-tau (●), K274R-tau (▴), and K274Q-tau (♦) were incubated with RNA for 24 h and the aggregation of tau was monitored by thioflavin S fluorescence (G,H) and 90° light scattering (I). The symbolic representation of tau without RNA is as follows: WT-tau (o), K274R-tau (Δ), and K274Q-tau (◊) (***P-value < 0.001). The results are represented as ±SEM.

The tau filaments formed by WT-tau and K274Q-tau were also observed by electron microscopy (Figure 6). In the absence of heparin, both WT-tau (Figure 6A) and K274Q-tau (Figure 6B) did not form any filaments. In the presence of heparin, WT-tau formed significantly shorter (P-value < 0.001) filaments (0.18 ± 0.01 µm) than the filaments (0.60 ± 0.02 µm) formed by K274Q-tau (Figure 6C,D), which further supported the finding that K274Q-tau has more aggregation propensity than WT-tau.

K274Q mutation in tau enhanced the filament formation.

Figure 6.
K274Q mutation in tau enhanced the filament formation.

Electron microscopic images of WT-tau (A) and K274Q-tau (B) incubated without heparin and WT-tau (C) and K274Q-tau (D) incubated with heparin. The scale bar is 0.2 µm.

Figure 6.
K274Q mutation in tau enhanced the filament formation.

Electron microscopic images of WT-tau (A) and K274Q-tau (B) incubated without heparin and WT-tau (C) and K274Q-tau (D) incubated with heparin. The scale bar is 0.2 µm.

The acetylation mimicking mutation at K274 enhanced the liquid–liquid phase separation of tau

During the aggregation, tau goes through the different intermediate steps like LLPS, oligomerization, and filamentation. A recent report suggested that the LLPS can initiate the aggregation of tau [42]. We examined the ability of K274Q-tau for the LLPS in comparison with WT-tau and K274R-tau. In the absence of PEG 8000 or heparin, tau failed to form droplets (Figure 7A,B). However, tau was found to form droplets in the presence of PEG 8000 or heparin indicating the LLPS of tau (Figure 7C–F). Furthermore, in the presence of both PEG 8000 and heparin, tau droplets were formed but the K274Q-tau droplets were bigger than the WT-tau droplets (Figure 7G,H). Heparin alone did not form droplets (Figure 7I). The quantification of WT-tau and K274Q-tau droplets indicated that K274Q-tau has more propensity to form droplets than WT-tau (Figure 7J). Furthermore, the phase diagram analysis indicated that the minimum concentration of PEG required for the phase separation of WT-tau and K274Q-tau was 5%. At 5% PEG, the minimum concentration of WT-tau and K274Q-tau required for the phase separation was determined to be 5 and 0.5 µM, respectively (Figure 7K,L). The finding indicated that the K274Q mutation decreases the critical concentration for the phase separation of tau.

The acetylation mimicking mutation at K274 enhanced the LLPS of tau.

Figure 7.
The acetylation mimicking mutation at K274 enhanced the LLPS of tau.

The tau (5 µM) was incubated with PEG 8000 and heparin for 2 h. Tau droplets were observed under a DIC microscope. Scale bar is 10 µm. The droplets marked by an arrow are shown as a magnified inset image. (A) WT-tau, (B) K274Q-tau, (C) WT-tau in the presence of 10% PEG 8000, (D) K274Q-tau in the presence of 10% PEG 8000, (E) WT-tau in the presence of heparin, (F) K274Q-tau in the presence of heparin, (G) WT-tau in the presence of both 10% PEG 8000 and heparin, (H) K274Q-tau in the presence of both 10% PEG 8000 and heparin, (I) only heparin. (J) The quantification of droplet formation of WT-tau and K274Q-tau in the presence of PEG or heparin or both PEG and heparin. The significance of data is validated by t-test (***P-value < 0.001, **P-value < 0.01). (K) Phase diagram of WT-tau. (L) Phase diagram of K274Q-tau.

Figure 7.
The acetylation mimicking mutation at K274 enhanced the LLPS of tau.

The tau (5 µM) was incubated with PEG 8000 and heparin for 2 h. Tau droplets were observed under a DIC microscope. Scale bar is 10 µm. The droplets marked by an arrow are shown as a magnified inset image. (A) WT-tau, (B) K274Q-tau, (C) WT-tau in the presence of 10% PEG 8000, (D) K274Q-tau in the presence of 10% PEG 8000, (E) WT-tau in the presence of heparin, (F) K274Q-tau in the presence of heparin, (G) WT-tau in the presence of both 10% PEG 8000 and heparin, (H) K274Q-tau in the presence of both 10% PEG 8000 and heparin, (I) only heparin. (J) The quantification of droplet formation of WT-tau and K274Q-tau in the presence of PEG or heparin or both PEG and heparin. The significance of data is validated by t-test (***P-value < 0.001, **P-value < 0.01). (K) Phase diagram of WT-tau. (L) Phase diagram of K274Q-tau.

The acetylation mimicking mutation at K274 enhanced the tau oligomerization

The oligomerization is an intermediate state of tau aggregation [41]. Among all forms of tau aggregates, the tau oligomers are most toxic [41]. Heparin was reported to induce both oligomerization and filamentation of tau depending on the duration of incubation [26,4345]. We also found that heparin-induced the formation of oligomers when tau was incubated with heparin for 6 h while filaments were formed when tau was incubated with heparin for 24 h. We checked the hydrodynamic diameter of tau by dynamic light scattering measurement. The size of both soluble WT-tau and K274Q-tau was found to be 5 ± 1 nm. In the presence of heparin, the size of WT-tau and K274Q-tau oligomers were determined to be 61 ± 16 and 311 ± 124 nm, respectively (Figure 8A), indicating that K274Q-tau formed significantly larger size oligomers than WT-tau (P-value < 0.05). The atomic force microscopic study revealed that K274Q-tau formed more oligomers than WT-tau in the presence of heparin (Figure 8B–E). Thus, the acetylation mimicking mutation at K274 residue of tau enhanced its oligomerization tendency. Furthermore, a dot blot assay using A11 oligomer-specific antibody showed that K274Q-tau had a higher tendency to form oligomers than WT-tau (Figure 8F). These results suggest that the acetylation mimicking mutation at K274 residue of WT-tau (4R2N tau) enhanced the oligomerization of tau.

An acetylation mimicking mutation at K274 residue of tau enhanced the oligomerization of tau.

Figure 8.
An acetylation mimicking mutation at K274 residue of tau enhanced the oligomerization of tau.

(A) The hydrodynamic diameter of soluble tau and oligomeric tau was determined by dynamic light scattering (n = 3, **P-value < 0.01). The results are represented as ±SEM. (BE) AFM images of tau oligomers. WT-tau incubated without heparin (B), K274Q-tau incubated without heparin (C), WT-tau incubated with heparin for 6 h (D), K274Q-tau incubated with heparin for 6 h (E). The scale bar is 2 µm. (F) The tau oligomerization checked with A11 oligomer-specific antibody. The loading control is 10% of the total sample and it was immuno-blotted with tau 5 antibody. The experiment was repeated three times.

Figure 8.
An acetylation mimicking mutation at K274 residue of tau enhanced the oligomerization of tau.

(A) The hydrodynamic diameter of soluble tau and oligomeric tau was determined by dynamic light scattering (n = 3, **P-value < 0.01). The results are represented as ±SEM. (BE) AFM images of tau oligomers. WT-tau incubated without heparin (B), K274Q-tau incubated without heparin (C), WT-tau incubated with heparin for 6 h (D), K274Q-tau incubated with heparin for 6 h (E). The scale bar is 2 µm. (F) The tau oligomerization checked with A11 oligomer-specific antibody. The loading control is 10% of the total sample and it was immuno-blotted with tau 5 antibody. The experiment was repeated three times.

The K274Q-tau oligomers were toxic to neuroblastoma cells

To investigate the neurotoxic effect of soluble WT-tau, K274R-tau, and K274Q-tau, SH-SY5Y cells were incubated with the tau proteins. The viability of the cells was found to reduce with increasing concentration of the tau variants indicating that soluble WT-tau, K274R-tau, and K274Q-tau were toxic to neuroblastoma cells at higher concentrations (Figure 9A). However, soluble K274Q-tau and WT-tau exerted similar cytotoxic effects on the neuroblastoma cells.

The oligomeric K274Q-tau was more toxic to SHYS5Y neuroblastoma cells WT-tau oligomers.

Figure 9.
The oligomeric K274Q-tau was more toxic to SHYS5Y neuroblastoma cells WT-tau oligomers.

(A,B) The soluble (A) or oligomeric (B) WT-tau, K274R-tau (KR) and K274Q-tau (KQ) were incubated with neuroblastoma cells for 48 h and the cytotoxicity was measured using alamar blue assay (n = 3, ns, non-significant, **P-value < 0.01). The results are represented as ±SD.

Figure 9.
The oligomeric K274Q-tau was more toxic to SHYS5Y neuroblastoma cells WT-tau oligomers.

(A,B) The soluble (A) or oligomeric (B) WT-tau, K274R-tau (KR) and K274Q-tau (KQ) were incubated with neuroblastoma cells for 48 h and the cytotoxicity was measured using alamar blue assay (n = 3, ns, non-significant, **P-value < 0.01). The results are represented as ±SD.

In a separate experiment, SH-SY5Y cells were grown in the absence and presence of oligomeric WT-tau, K274R-tau, and K274Q-tau. The viability of cells was determined to be 99 ± 1, 92 ± 6, and 68 ± 6% in the presence of 2, 3, and 5 µM of either oligomeric WT-tau and 97 ± 8, 91 ± 5, and 73 ± 7 in the presence of 2, 3, and 5 µM of oligomeric K274R-tau. On the other hand, the viability of the cells was determined to be 71 ± 6, 58 ± 10, and 49 ± 4% and in a presence of 2, 3, and 5 µM of K274Q-tau oligomers, respectively. The results indicated that the viability of SH-SY5Y cells was significantly lower in the presence of oligomers of K274Q-tau than WT-tau (P-value < 0.01) indicating that the oligomeric K274Q-tau was more toxic to neuroblastoma cells than the oligomeric WT-tau (Figure 9B). The higher toxicity of K274Q oligomers could be because of the higher penetration of K274Q oligomers into the cells or different conformation of K274Q-tau oligomers. The internalization of WT-tau and K274Q-tau oligomers into the neuroblastoma cells were examined using tau antibody and A11 antibody. The percentage of tau (tau-5 antibody) colocalizing with tau oligomers (A11) was estimated to be 86 ± 11%. Furthermore, Pearson's correlation coefficient of localization of tau oligomers (A11) with tau was determined to be 0.72 ± 0.09 (Supplementary Figure S1). The data indicate that tau oligomers were internalized into the cells.

Discussion

Tau acetylation is an important post-translational modification that plays a crucial role in tauopathies. In this study, we elucidated the role of acetylation mimicking mutation at K274 residue of tau on tubulin polymerization, tau aggregation, and neuronal toxicity. The K274Q mutation was found to alter the secondary structure of tau and it decreased the ability of tau to polymerize tubulin. Furthermore, WT-tau induced the bundling of microtubules in neuroblastoma cells while K274Q-tau did not induce microtubule bundling in these cells. In addition, the mutation (K274Q) enhanced the phase separation, oligomerization, and filament formation of tau. Furthermore, the oligomers of K274Q-tau produced more toxicity in neuroblastoma cells than that of WT-tau. The oligomeric tau was reported to spread into neurons by means of local and trans-synaptic mode, which results in a synaptic loss [41]. The synaptic oligomers are suggested to be internalized by receptor-mediated endocytosis [46], and dynein [47], actin and proteoglycan mediated micro-pinocytosis [48].

K274Q-tau regulate tubulin polymerization

Using SPR, we determined the dissociation constant for the interaction of tau and tubulin. Several techniques were used earlier to determine the binding of tau to microtubules. For example, the dissociation constant was determined to be 1.0 ± 0.5 µM by FRET analysis [49] and 280 nM ± 52 nM by sedimentation assay [50]. Our results together with the published literature show that tau has a very high affinity towards both dimeric tubulin and polymeric tubulin [49,50]. The K274Q mutation decreased the affinity of tau towards tubulin. Tau is known to bind to tubulin with its MTBR region [51]. The K274 residue lies in the R1 and R2 inter-repeat of tau. The R1 and R2 inter-repeat region (274KVQIINKKLDLSN286) of tau form a strong electrostatic interaction with microtubules and this region has twice the binding affinity with microtubules than any individual repeat [52]. The acetylation at K274 residue decreases the charge of the inter-repeat region, which may decrease the binding of tubulin with tau. The mutation (K274Q) induced structural changes in tau as indicated by far-UV circular dichroism, ANS fluorescence, and tryptophan fluorescence. The weakening of tau-tubulin interaction further decreases tubulin polymerization. Microtubules in the dendrites are more dynamic than the axon [53,54] indicating that the dynamic microtubules have a key role in the plasticity of the synaptic junction [53,54]. The decrease in tubulin polymerization can destabilize the microtubules and ultimately perturb neuronal plasticity. Furthermore, K274Q mutation reduced the binding affinity of tau to soluble tubulin; therefore, the acetylation at K274 residue of tau might increase the concentration of free tau in the cells leading to more tau aggregation and toxicity.

The acetylation mimicking mutation (K274Q) enhanced the aggregation of tau

In the physiological condition, the LLPS is a normal process of holding, condensing, and providing tau locally in axons where soluble and phase-separated tau are present in equilibrium [42]. Under the pathological condition, this equilibrium shifts to the phase separated tau, which further leads to the formation of aggregates [42]. The hyperacetylation of tau was suggested to disfavor the phase separation of tau [55]. In contrast, the acetylation mimicking mutation at the K274 position was found to favor the formation of tau droplets in the presence of molecular crowding agents like PEG 8000 and heparin. Nevertheless, the hyperacetylation of full tau decreases its pI from 8.2 to 5.5 [55] and, therefore, alters the electrostatic properties of tau while the point mutation at K274Q will not significantly change the pI.

Furthermore, the acetylation mimicking mutation at K274 residue of WT-tau (4R2N tau) enhanced the aggregation, oligomerization, and filamentation of tau. The Δ280 mutation found in the FTD, the acetylation mimicking mutation at K280 residue or the deletion of both K280 and K281 residue enhanced the aggregation of tau [17,56]. The findings indicated that the lysine residues of the inter-repeat region (274KVQIINKKLDLSN286) of tau have a crucial role in the tau aggregation [17,56]. Recently, pseudo-acetylation at K321, K274 residue of the 4R0N tau was shown to inhibit tau aggregation [57]. Interestingly, we found that the pseudo-acetylation at K274 residue of 4R2N tau enhanced the tau aggregation indicating that the acetylation mimicking mutation at K274 residue can produce different effects on different tau isoforms. This is supported by the reports that the tau isoforms with exon 2 and 10 facilitate the aggregation of tau [58,59] and 4R2N tau has the exon 2 while it lacks in 4R0N tau, therefore, the aggregation tendency of these isoforms can be different. The results also indicated that the acetylation of different isoforms of tau could be a possible mechanism for the regulation of tau aggregation inside cells and in various tauopathies.

Conclusion

The acetylation mimicking mutation (K274Q) induced structural changes in tau and reduced the ability of tau to polymerize tubulin. Furthermore, the K274Q mutation enhanced both the LLPS of tau and the aggregation of tau. In addition, the oligomers of K274-tau produced more toxicity in neuroblastoma cells than the oligomers of wild-type tau indicating that the acetylation at K274 residue of tau can regulate the functioning of tau.

Abbreviations

     
  • AD

    Alzheimer's disease

  •  
  • AMPAR

    α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

  •  
  • DIC

    differential interference contrast microscopy

  •  
  • FTD

    frontotemporal dementia

  •  
  • HRP

    horseradish peroxidase

  •  
  • KIBRA

    kidney and brain expressed protein

  •  
  • LC–MS-MS

    liquid chromatography with tandem mass spectrometry

  •  
  • LLPS

    liquid–liquid phase separation

  •  
  • MTBR

    microtubule-binding region

  •  
  • SPR

    surface plasmon resonance

Author Contribution

J.S.R. and A.K. performed the experiments. J.S.R., A.K., and D.P. analyzed the data and prepared the manuscript.

Funding

The work is supported by TATA Innovation Fellowship, Department of Biotechnology, Government of India (Grant No. BT/HRD/35/01/05/2017). J.S.R. is supported by INSPIRE Fellowship, Department of Science and Technology, Government of India.

Acknowledgements

We sincerely acknowledge Prof. S. Feinstein, University of California Santa Barbara for the 4R2N tau clone. We are also thankful to the Central Instrumentation Facility, Indian Institute of Technology Bombay for surface plasmon resonance, dynamic light scattering, atomic force microscopy, confocal microscopy, and electron microscopy facility.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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