The acetylation of tau inhibits its function and promotes pathological tau aggregation

Abstract

The microtubule associated protein tau promotes neuronal survival through binding and stabilization of MTs. Phosphorylation regulates tau-microtubule interactions and hyperphosphorylation contributes to the aberrant formation of insoluble tau aggregates in Alzheimer's disease (AD) and related tauopathies. However, other pathogenic post-translational tau modifications have not been well characterized. Here we demonstrate that tau acetylation inhibits tau function via impaired tau-microtubule interactions and promotes pathological tau aggregation. Mass spectrometry analysis identified specific lysine residues, including lysine 280 (K280) within the microtubule-binding motif as the major sites of tau acetylation. Immunohistochemical and biochemical studies of brains from tau transgenic mice and patients with AD and related tauopathies showed that acetylated tau pathology is specifically associated with insoluble, Thioflavin-positive tau aggregates. Thus, tau K280 acetylation in our studies was only detected in diseased tissue, suggesting it may have a role in pathological tau transformation. This study suggests that tau K280 acetylation is a potential target for drug discovery and biomarker development for AD and related tauopathies.

Conflict of interest statement

Competing financial interests: The authors declare no competing financial interests.

Figures

Figure 1. Tau acetylation impairs MT assembly and promotes tau fibrillization in vitro

(a) Recombinant full-length T40, 4R-tau MT-binding domain K18, or tau fibrils (see Methods for fibrillization assay) were acetylated by incubation with [14C]-labeled acetyl-CoA in the presence or absence of CBP. Reaction products were analysed by SDS–PAGE and Coomassie blue staining followed by overnight radiographic exposure using STORM phosphor-imager software. (b) T40 or K18 proteins were incubated with acetyl-CoA and/or recombinant CBP and reaction products were subjected to immunoblot analysis using a polyclonal anti-tau-specific antibody for MT repeat domain (E10) and an anti-acetyl-lysine antibody. (c) MT assembly activities of K18 proteins were evaluated in light-scattering assays. Tubulin monomers (30 μM) were mixed with 40 μM tau proteins in MT assembly buffer supplemented with 2 mM guanosine triphosphate. MT assembly was determined by monitoring absorbance every minute at 350 nm using a SpectraMax plate reader. Colour key is as follows: native unmodified K18 (pink squares), acetylated K18 (blue triangles), K18 containing K311D mutation (turquoise X’s) and no tau control (black diamonds). (d) Tau proteins (10 μM) were evaluated in fibrillization reactions using 10 μM heparin to induce assembly. At each time point, samples were incubated with 12.5 μM ThT and excitation/emission wavelengths were set to 450 and 510 nm, respectively. Colour key is as follows: native unmodified K18 (pink squares), acetylated K18 (blue diamonds) and K18-containing K311D mutation (turquoise triangles). Both MT assembly and fibrillization reactions were confirmed from n = 4 independent experiments. Error bars indicate standard error of the mean. (e) Tau proteins at indicated fibrillization time points (T = 0, 1, 3 h) were centrifuged at 100,000 g for 30 min to generate a pellet fraction (P) containing tau fibrils and supernatants (S) containing unassembled tau protein. Samples were analysed by SDS–PAGE and Coomassie staining to monitor fibril formation. (f) Four-hour time points from fibrillization reactions were analysed by negative-staining EM. Note, at the concentration of 10 μM, native K18 did not fibrillize, whereas acetylated K18 had fibrillized extensively with morphologies similar to AD-like PHFs. Scale bar, 200 nm.

Figure 2. Tau acetylation in cells inhibits tau-mediated MT stabilization

(a) Doxycycline (Dox)-treated HEK-T40 cells were cultured in the presence of either trichostatin A (TSA), nicotinamide (NCA) or both and pulse labelled with [3H]-acetate for 2 h. Tau was immunoprecipitated, treated with λ-phosphatase where indicated, and analysed by SDS–PAGE followed by autoradiography and exposure to film for 2 weeks. Total cellular lysates were immunoblotted using anti-tau (T14/T46), PHF-1 and GAPDH antibodies. (b) Cells transfected with CBP were treated with TSA and acetylated tau levels were determined by immunoprecipitation/immunoblot analysis using anti-acetylated lysine antibody. Cellular lysates were immunoblotted using T14/46, PHF-1, AT8 and GAPDH antibodies. (c) Cells were transfected with CBP and either wild-type (WT) HDAC6 or the catalytically dead (CD) HDAC6-H611A mutant and tau acetylation was determined by immunoblotting similar to b above. (d) QBI-293 cells were transfected with WT T40, 2KR (K280/281R), 3KR (K163/280/281R) or 4KR (K163/280/281/369R) mutant T40 and cell lysates were immunoblotted using anti-acetyl-lysine and total tau (T14/T46) antibodies. (e–j) QBI-293 cells were transfected with WT T40 (e, red) and MT bundling was determined by immunofluorescence using anti-acetylated tubulin antibodies as shown for WT (f, green). Merged images indicate stabilized microtubules as shown for WT (g). QBI-293 cells were transfected with 4KQ (K163/280/281/369Q) (h, red) and MT bundling was determined by immunofluorescence using anti-acetylated tubulin antibodies as shown for 4KQ (i, green) and merged images (j). Representative images are shown from n = 4 independent experiments. Note, WT tau (e–g), but not 4KQ (h–j), promotes robust MT bundling. Scale bar, 50 μm. (k) QBI-293 cells were transfected with WT T40 (red bar), K280Q (green bar), 4KQ (K163/280/281/369Q) (blue bar) or 4KR (purple bar) mutant plasmids and MT bundling was determined from tau-expressing cells by quantification of ten fields/transfection and values are represented as %MT bundling from tau-expressing cells. Error bars indicate standard error of the mean. *P value = 0.006, ***P value = 8.46×10− 6 as determined by Student’s t-test.

Figure 3. Tau acetylation in Tg mouse models with tau pathology

(a–f) Immunohistochemistry (IHC) was used to analyse cortical sections from the following lines of mice: wild-type (WT) mice using anti-ac-K280 (a) or AT8 (b) antibodies, PS19 monogenic mice using anti-ac-K280 (c) or AT8 antibodies (d) and PS19;PDAPP bigenic mice using anti-ac-K280 (e) and AT8 antibodies (f). (g–i) Double-labelling immunofluorescence microscopy using anti-ac-K280 (g, red) and AT8 (h, green) antibodies on hippocampal sections from PS19;PDAPP mice showed extensive co-localization of acetylated and hyperphosphorylated tau inclusions (merged image, i). (j–l) Double labelling using anti-ac-K280 (j, red) and ThS (k, green), an amyloid-binding dye, also demonstrated co-localization of acetylated tau with Thioflavin-S-positive NFTs (merged image, l). Scale bars, 100 μm. (m, n) Sequential biochemical fractionation was performed on WT, PS19 and PS19/PDAPP mouse cortices using RIPA buffer followed by urea extraction. Soluble (m) and insoluble (n) extracted tau proteins were analysed by western blotting using anti-ac-K280, PHF-1 and total tau (T14/T46) antibodies.

Figure 4. Tau acetylation is associated with tau aggregation in human tauopathies

(a–f) Immunohistochemistry (IHC) was used to analyse cortical sections from the following tauopathies: Alzheimer’s disease (AD) using anti-ac-K280 (a) or PHF-1 (b) antibodies, corticobasal degeneration (CBD) using anti-ac-K280 (c) or PHF-1 (d) antibodies and Pick’s disease (PiD) using anti-ac-K280 (e) or PHF-1 (f) antibodies. Shown are representative images of tau inclusions with anti-ac-K280 and PHF-1 immunoreactivity. AD and 4R-tau predominant tauopathies (for example, corticobasal degeneration) were ac-K280 immunopositive (a, c), whereas 3R-tau predominant Pick’s disease was negative (e). (g–l) Double-labelling immunfluorescence was performed on cortical sections from AD using anti-ac-K280 (g, red) and PHF-1 (h, green) antibodies (merged image, i). Similar analysis was performed using sections from CBD using anti- ac-K280 (j, red) and PHF-1 (k, green) antibodies (merged image, l). (m–o) Double labelling of neurofibrillary tangles (NFTs) in AD cortex was detected using anti-ac-K280 (m, red) and Thioflavin S (n, green) and the corresponding merged image is shown in o. Insets represent ×60 magnification. Scale bar, 50 μm. (p) Biochemical isolation of enriched PHFs was performed on frontal cortex from indicated tauopathies and solubilized PHF-tau was analysed by immunoblotting using anti-ac-K280, PHF-1 and total tau (T14/T46) antibodies. Ac-K280 and PHF-1 immunoreactivity was quantified using Multi Guage v2.3 and raw intensities are represented below as percent modified tau species/total tau intensities to compare acetylated versus phosphorylated tau ratios.